carbanion in water

7/30/2019 CARBANION IN WATER

1/26

FormationandStabilityofCarbocations andCarbanions inWater andIntrinsic Barriers toTheir Reactions

JOHN P. RICHARD,* TINA L. AMYES, ANDMARIA M. TOTEVA

Department of Chemistry, University at Buffalo,State University of New York,Buffalo, New York 14260-3000

Received J uly 26, 2001

ABSTRACTLifetimes in water as short as 10-11 s have been determined forcarbocations and carbanions by referencing the rate of theirreaction with solvent species to that for the appropriate clockreaction, and equilibrium constants have been determined as theratio of rate constants for their formation and breakdown. Rate-

equilibrium correlations for these organic ions are often poor andsometimes even defy the simple generalization that reactivityincreases with decreasing stability. This seemingly confusing bodyof data can be understood through consideration of the both theMarcus intrinsic barrier and the thermodynamic driving force toreaction of these organic ions.

IntroductionThe generation of most localized and many delocalized

carbanions and carbocations in water is problematic,

because the chemical barrier to their Brnsted base or

Lewis acid reactions with solvent is often smaller than that

for a bond vibration.1

An important challenge to laboratorychemists and living systems is the development of meth-

odology for cleavage and synthesis of carbon-carbon

bonds through carbanion or carbocation intermediates.

The formation of these species is often facilitated by

delocalization of charge away from carbon and onto

distant electronegative atoms. However, these ions are

referred to as carbocations and carbanions even when the

bulk of the charge lies distant from the reacting carbon,

because the preponderance of their chemical reactions

occur at carbon. Such resonance-stabilized carbocations

and carbanions are ubiquitous intermediates of metabolic

and synthetic organic reactions, and the study of their

chemical reactivity is of interest to both organic chemists

and biochemists.

Only a few laboratories are investigating the reactionsof carbocations and carbanions in aqueous solution. The

restricted interest in these important reactions has pro-

vided motivation for the following work described in this

Account. (1) Development of experimental methods to

obtain reliable estimates of lifetimes, down to the pico-

second regime, of carbocations and carbanions in water,

which are intermediates of chemical and biochemical

reactions. (2) Characterization of the dynamics of car-

bocation-anion ion pair intermediates of solvolysis reac-

tions. (3) Determination of equilibrium constants for

formation of carbocations and carbanions. (4) Rational-

ization of the changes in these rate and equilibrium

constants that occur with variations in substrate structure.

ReactionClocksMany sophisticated studies of reactive intermediates have

focused on determination of absolute rate constants by

directly monitoring the disappearance of the intermediate

generated by laser flash photolysis.2We have used simpler,

but effective, indirect methods which reference the un-

known rate constant for reaction of the intermediate with

solvent water to that for a second reaction which serves

as a clock. The rate constants for these clock reactions

are known with good precision, because they are limited

by the rate constant for a physical transport step which

is largely independent of the structure of the intermediate.

Clocks for Carbocations. There are sharp decreases in

the rate constant ratio kaz/ks (M-1) for partitioning of

1-arylethyl and 1-aryl-2,2,2-trifluoroethyl carbocations

between nucleophilic addition of azide ion, kaz (M-1 s-1,

Scheme 1), and solvent, ks (s-1), when the carbocation is

destabilized by the addition of electron-withdrawing

aromatic ring substituents.3,4

This is because kaz is theinvariant rate constant for a diffusion-limited reaction, but

ks is limited by the chemical barrier to solvent addition

and increases with decreasing carbocation stability. Values

ofks for reaction of a variety of carbocations with aqueous

solvents have been obtained from kaz/ks (M-1) determined

by product analysis (eq 1), using kaz ) 5 109 M-1 s-1

(Scheme 1).3,4 In cases where a comparison is possible,

provided kaz/ks e104 M-1, the values ofks from this azide

* Phone: (716) 645-6800 ext 2194. Fax: (716) 645-6963. E-mail:[email protected].

J ohn P. Richard receiveda B.S. degree inbiochemistryand his Ph.D. degree inchemistry (1979) from The Ohio State University. He is currently Professor ofChemistry at the University at Buffalo, SUNY.

Tina L. Amyes receivedB.A. and M.A. degreesin natural sciences and her Ph.D.degree in chemistry (1986) from the University of Cambridge (England). She iscurrently an Adjunct Assistant Professor at the University at Buffalo, SUNY.

Maria M. Toteva received B.S. and M.S. degrees in chemistry from SofiaUniversity(Bulgaria) andherPh.D.degree in chemistry(1999) fromthe UniversityatBuffalo, SUNY. She is currently a Senior Scientist at the University at Buffalo,SUNY.

Scheme 1

Acc. Chem. Res. 2001, 34, 981-988

10.1021/ar0000556 CCC: $20.00 2001 American Chemical Society VOL. 34, NO. 12, 2001 / ACCOUNTS OF CHEMICAL RESEARCH 981Published on Web 10/24/2001


2/26

ion clock are in excellent agreement ((20%) with those

determined directly by generation of the same carbocation

by laser flash photolysis.2a,5

Absolute rate constants for carbocation-nucleophile

addition have been used to calculate equilibrium con-

stants for carbocation formation. (1) Values ofks (s-1) have

been combined with rate constants kH (M-1 s-1) for acid-

catalyzed cleavage of the corresponding alcohols to give

values of pKR for R-substituted benzyl, benzhydryl, and

9-fluorenyl carbocations (eq 3, Scheme 1).6 (2) Rate

constants ksolv (s-1) for the stepwise solvolysis of gem-

diazides, R-alkoxy azides, and R-substituted benzylic

azides through carbocation intermediates have been

determined and combined with kaz

) 5 109 M-1 s-1 for

the diffusion-controlled capture of these carbocations by

azide ion to give equilibrium constants Kaz ) ksolv/kaz (M)

for formation of the carbocation from the neutral azide

ion adduct (Scheme 1).7

Clocks for Ion Pairs. The estimated value of k-d ) 1.6

1010 s-1 for diffusional separation of carbocation-anion

ion pairs to give the free ions in 50:50 (v/v) water/

trifluoroethanol 8 serves as a useful clock for other reac-

tions of ion pairs. We have used this clock to determine a

number of rate constants that describe the dynamics of

these short-lived intermediates.

(1) Reaction of 1-(4-thiomethylphenyl)ethyl thionoben-

zoate (1, X ) S, Y ) O, Z ) MeS, Ar ) C6H5) in 50:50 (v/v)

water/trifluoroethanol gives 84% of the rearranged thiol-

benzoate ester 2 and 14% of the solvent adducts 3

(Scheme 2).9 It was shown that kc > k-r and k-d . ks, so

that the product ratio [2]/[3] ) 84/16 corresponds to kr/

k-d ) 6 for partitioning of the ion pair intermediate

between reorganization that exchanges the positions of

O and S at the leaving group anion and diffusional

separation to give the free ions.9 This was combined with

k-d ) 1.6 1010 s-1 to give kr ) 1011 s-1 as the absolute

rate constant for reorganization of the ion pair intermedi-

ate within an aqueous solvation shell (Scheme 2).9

(2) The rate constant ratios for partitioning of the ion

pair intermediate generated by cleavage of 4-MeC6H4-13CH(Me)18OC(16O)C6F5 (1, X ) 16O, Y ) 18O, Z ) Me, Ar )

C6F5) between diffusional separation (k-d) and direct

nucleophilic addition of solvent (ks) to give 3, and internal

return with 18O scrambling (kc) to give the isomerization

product 2, have been determined and combined with the

known values of k-d and ks to give kc ) 7 109 s-1 (kc |-0.5| for unfavorable proton

transfer. This curvature is predicted by different forms of

the Marcus equation, but the data in Figure 1 do not show

satisfactory fits to these models.13 Much of the change in

the thermodynamic driving force for proton transfer that

occurs with increasing pKa of these R-carbonyl carbon

acids is due to a decrease in the resonance stabilization

of the enolate product, which is also expected to lead to

a decrease in the Marcus intrinsic barrier for proton

transfer.23 We have therefore proposed that the change

to a steeper, more negative, slope of the correlation in

Figure 1 with increasing thermodynamic barrier to carbon

deprotonation that is predicted by the Marcus equation

is masked by opposing increases in kHO resulting from

concomitant decreases in .13

The intrinsic barrier for protonation of R-cyano car-

banions is substantially smaller than that for protonation

of enolates and other resonance-stabilized carbanions,36

but there has been little consideration of the broader

implication of this small barrier. We have found it useful

to compare C-protonation of the cyanomethyl carbanion

(Scheme 8) with O-protonation of acetate ion.15 The small

intrinsic barrier to O-protonation of acetate ion reflects

the small stabilization associated with delocalization of

charge between the two oxygens.37 There is a similar small

intrinsic barrier associated with C-protonation of R-cyano

carbanions (kHOH, Scheme 8),11 where the charge is

distributed approximately equally between carbon (60%)and the R-cyano group (40%)38 and the stabilization

associated with charge delocalization is presumablysmall.

Absolute andRelative Magnitudes of IntrinsicBarriers to Organic Reactions

We have determined absolute and relative intrinsic bar-

riers for several organic reactions and have offered

sensible interpretations of these data. These determina-tions are limited; our interpretations are complex and

undergoing constant evaluation. Input from the organic

community would be valued.

Nucleophile Addition to a Quinone Methide. The

reactivity of the quinone methide 10 toward nucleophiles

is similar to that of other strongly resonance-stabilized

carbocations.22,39 There is no simple relationship between

the rate and equilibrium constants Keq ) kNu/ksolv (M-1)

for addition of halide and acetate ions to 10 (Scheme 9),

because there are differences of up to 7 kcal/mol in the

Marcus intrinsic barrier for addition of different nu-

cleophiles: Nu-, (kcal/mol) I-, 12.4; Br-, 13.9; Cl-, 15.4;

AcO-, 19.8.22 Similar differences in are observed foraddition of halide ions to the trityl carbocation. 22

The R-deuterium isotope effect for concerted bimo-

lecular nucleophilic substitution at N-(methoxymethyl)-

Table 1. Effects of Strongly E lectron-Donatingr-Substituents on the Thermodynamic Stability of

Primary Alkyl C arbocations and on the RateConstant for Nucleophilic Addition of Solvent Water

(Scheme 1)a

a In water at 25 C. bGaz ) -RT ln Kaz (Scheme 1). Valuesof K

az) k

solv/k

azwere taken from ref 34. c Rate constant for

nucleophili c additi on of solvent water taken from ref 34.

Scheme 8

Scheme 9

Carbocations and Carbanions in Water Richard et al.

986 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 12, 2001


7/26

N,N-dimethylanilinium ions decreases with increasing

hardness of the nucleophile: Nu-, kH/kD I-, 1.18; Br-,

1.16; Cl-, 1.13; AcO-, 1.07.40 Therefore, the hybridization

of the central carbon in the transition state for nucleo-

philic substitution by iodide ion is close to that for the

methoxymethyl carbocation (MeOCH2+), and there is a

shift to a transition state with greater bonding between

the nucleophile and the central carbon on moving along

the series I-, Br-, Cl-, AcO-. These results suggest that

the transition state for nucleophile addition to 10 and the

trityl carbocation is reached earlier (more cationic), and

with a lower intrinsic barrier, for soft strongly polarizable

nucleophiles such as iodide ion compared with hard

weakly polarizable nucleophiles such as acetate ion,

because the former nucleophiles provide greater orbital

overlap at longer bond distances.22 If different degrees of

bond formation are observed at the transition state for

thermoneutral addition of different nucleophiles to 10,

then the intersecting curves that constitute the reaction

coordinate profile for nucleophile addition must in some

cases exhibit pronounced asymmetry (Scheme 10).

We have examined the addition of thiols and sulfides

to 10.27 The polarizable methyl group at dimethyl sulfide

provides significant stabilization of the charge at the

cationic sulfur of the nucleophile addition product, but itcreates an even larger destabilizing steric/electrostatic

interaction with the R-CF3 groups. The reaction proceeds

through a transition state at which there is an imbalance

between the large expression of the stabilizing interactions

of the polarizable methyl group and the smaller expression

of destabilizing steric/electrostatic interactions.27 These

data suggest that bonding interactions between 10 and

Me2S in the transition state for nucleophile addition are

expressed at a large distance, which minimizes steric/

electrostatic interactions between the nucleophile and the

R-CF3 groups, so that these destabilizing interactions

develop after the transition state has been traversed on

the reaction coordinate.Reactions of Oxocarbenium Ions. The intrinsic barrier

for nucleophilic addition of methanol to the oxocarbe-

nium ion 11H+ to give 12H+, MeOH ) 6.5 kcal/mol, is

about 50% smaller than that for deprotonation of 11H+

at carbon by solvent water to give 11, p ) 13.8 kcal/mol

(Scheme 11).41 The difference in these intrinsic barriers

may be related to the requirement for more extensive

electronic reorganization on proceeding to the transition

state for deprotonation of 11H+ compared with the

transition state for direct nucleophile addition to the

carbocation.41

Aldol Addition Reactions. The requirement for rehy-

bridization and electronic reorganization at the benz-

aldehyde carbonyl group upon formation of the tetra-

hedral adduct with hydroxide ion results in an 8 kcal/mol

larger Marcus intrinsic barrier compared with that for

direct proton transfer between hydroxide ion and water.42

Similarly then, the intrinsic barrier for addition of an

enolate ion to the benzaldehyde carbonyl group (kc,

Scheme 12) might also be expected to be larger than that

for protonation of the enolate by solvent water (kHOH,

Scheme 12). In fact, we have shown that these processes

have very similar Marcus intrinsic barriers of ) 14.1

kcal/mol for kc and ) 14.7 kcal/mol for kHOH.42 Thissuggests that there may be compensating stabilization of

the transition state for aldol addition by favorable interac-

tions between the HOMO of the soft enolate nucleophile

and the LUMO of the soft carbonyl group electrophile.42

This work is the result of the training that J.P.R. and T.L.A.

received as Postdoctoral Fellows in the laboratory of William P.

Jencks at Brandeis University and the efforts of their co-workers

at Kentucky and Buffalo. Our work has been generously supported

by Grant GM39754 from the National Institutes of Health.

References(1) J encks, W. P. How Does a Reaction Chooseits Mechanism? Chem.

Soc. Rev. 1981, 10, 345-375.(2) (a) McClelland, R. A. Flash Photolysis Generation and Reactivities

of Carbenium Ions and Nitrenium Ions. Tetrahedron 1996, 52,6823-6858. (b) Chiang,Y .;Kresge, A. J . Enols and Other ReactiveSpecies. Science1991, 253, 395-400.

(3) Richard, J . P.; Rothenberg, M . E.; J encks, W. P. Formation andStability of Ring-Substituted 1-Phenylethyl Carbocations.J . Am.Chem. Soc. 1984, 106, 1361-1372.

(4) Richard, J . P. The Extraordinarily Long Lifetimes and OtherProperties of Highly Destabilized Ring-Substituted 1-Phenyl-2,2,2-trifluoroethyl Carbocations.J . Am. Chem. Soc.1989, 111, 1455-1465.

(5) McClelland, R. A.; Cozens, F. L.; Steenken, S .; A myes, T. L.;Richard, J . P. Direct Observation of-Fluoro-substituted 4-Meth-oxyphenethyl Cations by Laser Flash Photolysis. J . Chem. Soc.,Perkin Trans. 21993, 1717-1722.

Scheme 10

Scheme 11

Scheme 12


VOL. 34, NO. 12, 2001 / ACCOUNTS OF CHEMICAL RESEARCH 987


8/26

(6) Amyes, T. L.; Richard, J . P.; Novak, M. Experiments and Calcula-tions for Determination of the Stabilities of Benzyl, Benzhydryl,and Fluorenyl Carbocations: Antiaromaticity Revisited. J . Am.Chem. Soc. 1992, 114, 8032-8041.

(7) A myes, T. L.; Stevens, I. W.; Richard, J . P. The Effects ofR-Substituents on the Kinetic and Thermodynamic Stability of4-Methoxybenzyl Carbocations: Carbocation Lifetimes That AreIndependent of Their Thermodynamic Stability. J . Org. Chem.1993, 58, 6057-6066.

(8) Richard, J . P.; J encks, W. P. Reactions of Substituted 1-PhenylethylCarbocations with Alcohols and Other Nucleophilic Reagents.J .Am. Chem. Soc. 1984, 106, 1373-1383.

(9) Richard, J . P.; Tsuji, Y. Dynamics for Reaction of an Ion Pair inAqueous Solution: The RateConstant for Ion Pair Reorganization.J . Am. Chem. Soc. 2000, 122, 3963-3964.

(10) Tsuji, Y.; M ori, T.; Richard, J . P.; Amyes, T. L.; Fujio, M .; Tsuno,Y. Dynamics for Reaction of an Ion Pair in Aqueous Solution:Reactivity of Carboxylate Anions in Bimolecular Carbocation-Nucleophile Addition and Unimolecular Ion Pair Collapse. Org.Lett. 2001, 3, 1237-1240.

(11) Richard, J . P.; Williams, G.; Gao, J . Experimental and Computa-tional Determination of the Effect of theCyano Group on CarbonAcidity in Water. J . Am. Chem. Soc. 1999, 121, 715-726.

(12) Rios, A.; A myes, T. L.; Richard, J . P. Formation and Stability ofOrganic Zwitterions in Aqueous Solution: Enolates of theA minoGlycine and Its Derivatives.J . Am. Chem. Soc.2000, 122, 9373-9385.

(13) Amyes, T. L.; Richard, J . P. Determination of the pKa of EthylAcetate: Brnsted Correlation for Deprotonation of a SimpleOxygen Ester in Aqueous Solution.J . Am. Chem. Soc.1996, 118,3129-3141.

(14) Richard, J . P .; Williams, G.; ODonoghue, A. C.; Amyes, T. L.Formation and Stability of Enolates of Acetamide and AcetateIon: An Eigen Plot for Proton Transfer at R-Carbonyl Carbon. J .Am. Chem. Soc., submitted for publication.

(15) Eigen, M. Proton Transfer, Acid-Base Catalysis, and EnzymaticHydrolysis. Angew. Chem., Int. Ed. Engl. 1964, 3, 1-72.

(16) Keeffe,J . R.; Kresge,A. J . In TheChemistry of Enols; Rappoport,Z., Ed.; J ohn Wiley and S ons: Chichester, 1990; pp 399-480.

(17) Amyes, T. L.; Richard, J . P. Generation and Stability of a SimpleThiol Ester Enolatein Aqueous Solution.J . Am. Chem. Soc.1992,114, 10297-10302.

(18) Rios, A .; Richard, J . P. Biological Enolates: Generation andStability of the Enolate of N-Protonated Glycine Methyl Ester inWater. J . Am. Chem. Soc. 1997, 119, 8375-8376.

(19) Malhotra, S. K.;Ringold, H. J . Chemistry of Conjugate Anions andEnols. IV. The Kinetically Controlled Enolization of R,-Unsatur-ated Ketones and the Natureof the Transition State.J . Am. Chem.Soc. 1964, 86, 1997-2003.

(20) M arcus, R. A. Theoretical Relations among Rate Constants,Barrier, and Brnsted S lopes of Chemical Reactions. J . Phys.Chem. 1968, 72, 891-899.

(21) Marcus, R. A. Unusual Slopes of Free Energy Plots in Kinetics.J .Am. Chem. Soc. 1969, 91, 7224-7225.

(22) Richard, J . P.; Toteva, M. M.; Crugeiras, J . Structure-ReactivityRelationships and Intrinsic Reaction Barriers for NucleophileAdditions to a Quinone Methide: A Strongly Resonance-Stabilized Carbocation.J . Am. Chem. Soc.2000, 122, 1664-1674.

(23) Bernasconi, C. F. The Principle of N onperfect Synchronization:More Than a Qualitative Concept? Acc.Chem. Res.1992, 25, 9-16.

(24) Kresge, A. J . The N itroalkane Anomaly. Can. J . Chem. 1974, 52,1897-1903.

(25) Amyes, T. L.; J encks, W. P. Lifetimes of Oxocarbenium Ions inAqueous Solution from Common Ion Inhibition of the SolvolysisofR-Azido Ethers by Added Azide Ion. J . Am. Chem. Soc. 1989,111, 7888-7900.

(26) Richard, J . P.; A myes, T. L.; Rice, D. J . Effects of ElectronicGeminal Interactions on the Solvolytic Reactivity of Methoxym-ethyl Derivatives. J . Am. Chem. Soc. 1993, 115, 2523-2524.

(27) Toteva, M. M.; Richard, J . P. Structure-Reactivity Relationshipsfor Addition of Sulfur N ucleophiles to Electrophilic Carbon:Resonance, Polarization, and Steric/Electrostatic Effects. J . Am.Chem. Soc. 2000, 122, 11073-11083.

(28) Richard, J . P. A Consideration of the Barrier for Carbocation-Nucleophile Combination Reactions. Tetrahedron1995, 51, 1535-1573.

(29) Richard, J . P.; Amyes, T. L.; Bei, L.; Stubblefield, V. Effect of-Fluorine Substituents on the Rate and Equilibrium Constants

for the Reactions ofR-Substituted 4-Methoxybenzyl Carbocationsand on theReactivity of a SimpleQuinone Methide.J . Am. Chem.Soc. 1990, 112, 9513-9519.

(30) Richard, J . P.; Amyes, T. L.; Stevens, I. W. Generation andDetermination of the Lifetime of an R-Carbonyl SubstitutedCarbocation. Tetrahedron Lett. 1991, 32, 4255-4258.

(31) Richard,J . P.;Lin, S.-S.;Buccigross, J . M.; Amyes, T. L. How DoesOrganic S tructure Determine Organic Reactivity? NucleophilicSubstitution and Alkene-Forming Elimination Reactions ofR-Car-bonyl and R-Thiocarbonyl Substituted Benzyl Derivatives.J . Am.Chem. Soc. 1996, 118, 12603-12613.

(32) Lee, I.; Chung D. S.; J ung, J . H. Theoretical Studies of the EffectsofR-Substituents on the Resonance Demand of 4-MethoxybenzylCarbocations. Tetrahedron1994, 50, 7981-7986.

(33) J agannadham, V.; Amyes, T. L.; Richard, J . P. Kinetic andThermodynamic Stabilities ofR-Oxygen- and R-Sulfur-StabilizedCarbocations in Solution. J . Am. Chem. Soc. 1993, 115, 8465-8466.

(34) Richard, J . P.; A myes, T. L.; J agannadham, V.; Lee, Y .-G.; Rice,D. J . Spontaneous Cleavage of gem-Diazides: A Comparison ofthe Effects of R-Azido and Other Electron-Donating Groups onthe Kinetic and Thermodynamic Stability of Benzyl and A lkylCarbocations in Aqueous Solution.J . Am. Chem. Soc.1995, 117,5198-5205.

(35) Schleyer, P. v. R. A Systematic Survey of the Effects of Substit-uents on the Structures and Energies of the Principal OrganicReaction Intermediates. Pure Appl. Chem. 1987, 59, 1647-1660.

(36) Hojatti, M.; Kresge, A. J .; Wang, W.-H. Cyanocarbon Acids: DirectEvidence That Their Ionization Is Not an Encounter-ControlledProcess and Rationalization of the U nusual Solvent IsotopeEffects. J . Am. Chem. Soc. 1987, 109, 4023-4028.

(37) Rablen, P. R. Is the Acetate A nion S tabilized by Resonance orElectrostatics? A Systematic Structural Comparison.J . Am. Chem.Soc. 2000, 122, 357-368.

(38) Wiberg, K. B.; Castejon, H. Carbanions 2. Intramolecular Interac-tions in Carbanions Stabilized by Carbonyl, Cyano, Isocyano, and

Nitro Groups. J . Org. Chem.1995

, 60, 6327-

6334.(39) Richard, J . P. Mechanisms for theU ncatalyzed and Hydrogen IonCatalyzed Reactions of a Simple Quinone Methide with Solventand Halide Ions. J . Am. Chem. Soc. 1991, 113, 4588-4595.

(40) Knier, B. L.; J encks, W. P. M echanism of Reactions of N-(Meth-oxymethyl)-N, N-Dimethylanilinium Ions w ith Nucleophilic Re-agents. J . Am. Chem. Soc. 1980, 102, 6789-6798.

(41) Richard, J . P.; Williams, K. B.; Amyes, T. L. Intrinsic Barriers forthe Reactions of an Oxocarbenium Ion in Water. J . Am. Chem.Soc. 1999, 121, 8403-8404.

(42) Richard, J . P.; Nagorski, R. W. Mechanistic Imperatives forCatalysis of Aldol Addition Reactions: Partitioning of theEnolateIntermediate between Reaction with Brnsted Acids and theCarbonyl Group. J . Am. Chem. Soc. 1999, 121, 4763-4770.

AR0000556


988 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 12, 2001


9/26

Phosphate Binding Energy and Catalysis bySmall and Large Molecules

JANET R. MORROW, TINA L. AMYES, AND JOHN P. RICHARD*Department of Chemistry, University at Buffalo, State University of New York,

Buffalo, New York 14260-3000

RECEIVED ON SEPTEMBER 11, 2007

C O N S P E C T U S

Catalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the differ-ence between the activation barriers for the uncatalyzed (GHOq) and catalyzed (GMeq) reactions, which corresponds to thebinding energy (GS

q) GMe

q- GHO

q) for transfer of the reaction transition state from solution to the catalyst. This transi-

tion state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding

of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between

low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound sub-

strate. Work on catalysis by small molecules is exemplified by studies on the mechanism of action of Zn2(1)(H2O). A binding energy

ofGSq

) -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate pro-

file for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form

[Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygen-

ionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high affinity for oxyphosphorane transition state

dianions and a stable methyl phosphate transition state analogue, compared with the affinity for phosphate monoanion sub-

strates. The transition state binding energies, GSq, for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion

complexes reflect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of GSq

are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with GSq observed

for catalysis by free metal ions, where the ligands are water. Enzymes are unique in having evolved mechanisms to effectively

utilize binding interactions with nonreacting fragments of the substrate in stabilization of the reaction transition state. Orotidine5-monophosphate decarboxylase, R-glycerol phosphate dehydrogenase, and triosephosphate isomerase catalyze dissimilar decar-

boxylation, hydride transfer, and proton transfer reactions, respectively. Each enzyme derives ca. 12 kcal/mol of transition state

stabilization from protein interactions with the nonreacting phosphate group, which is larger than the highest 10 kcal/mol tran-

sition state stabilization that we have determined for small-molecule catalysis of phosphate diester cleavage in water. Each of these

enzymes catalyze the slow reaction of a truncated substrate that lacks the phosphate group, and in each case, the reaction of the

truncated substrate is strongly activated by the allostericbinding of the second substrate piece phosphite dianion, HPO32-. We

propose a modular design for these enzymes with a classical active site that recognizes the reactive substrate fragment and a sep-

arate phosphodianion binding site. The second site is created, in part, by flexible protein loops that wrap around the substrate

phosphodianion group and burythe substrate in an environment with an optimal local dielectric constant for the catalyzed reac-

tion and with the most favorable positioning of the catalytic side chains. This design is easily generalized to a wide variety of

enzyme-catalyzed reactions.

Vol. 41, No. 4 April 2008 539-548 ACCOUNTS OF CHEMICAL RESEARCH 539Published on the Web 02/23/2008 www.pubs.acs.org/acr10.1021/ar7002013 CCC: $40.75 2008 American Chemical Society


10/26

Introduction

Linus Pauling noted that catalysis in general, and by enzymes

in particular, will result from the development of tight inter-

actions between the catalyst and the transition state for the

catalyzed reaction.1 This is shown by the transition state bind-

ing energy, GSq (Scheme 1). Catalysis by most enzymes is so

efficient that release of products would be strongly rate-de-

termining if they were to bind with the same affinity as the

transition state. Consequently, enzymes show a large discrim-

ination and bind their substrates/products much more weakly

than the reaction transition state (Scheme 1).2

The Pauling paradigm has a particular appeal when trying

to explain catalysis to students. On the other hand, there are

issues that must be addressed if this model is to be accepted

as a starting framework for explaining enzyme catalysis. Theseinclude the following:

(1)Recently, Zhang and Houk wrote: While complementar-

ity of the type proposed by Pauling can account for accel-

eration up to 11 orders of magnitude, most enzymes

exceed that proficiency.3 They therefore proposed that

enzymes achieve over 15 kcal/mol oftransition state bind-

ing not merely by a concatenation of noncovalent effects

but by covalent bond formation between enzyme or cofac-

tor and transition state, involving a change in mechanism

from that in aqueous solution.3

(2)There are only small differences in the structure of the sub-

strate/product and the transition state for many enzyme-

catalyzed reactions. The origin of the large differential

binding of these species is generally unknown.2

(3)Enzymes are large molecules that undergo many types of

motion, some of which may be coupled to catalysis. Fur-

ther, catalysis of chemical reactions must ultimately be

explained at the level of quantum mechanics. Since nei-

ther protein dynamics nor quantum mechanics are central

to the Pauling paradigm, more sophisticated models forcatalysis might either use this paradigm as a starting point

or as a specialized example or, in the most extreme case,

discard it entirely.

Our goal as experimentalists has been to characterize the

mechanisms for catalysis by small metal ion complexes and

by larger enzymes where much or all of the catalytic power is

derived from the utilization of phosphate binding energy. We

summarize here the results of this work and provide an inter-

pretation of our results within the framework of the Pauling

model.

Phosphate Diester Cleavage

The design of catalysts to cleave RNA has considerable intel-

lectual appeal, along with potential to produce RNA cleavage

reagents that have practical applications.46J. R. Morrow and

co-workers have shown that lanthanide(III) complexes are effi-cient catalysts of the cleavage of RNA,7,8 and they have exam-

ined cleavage of the 5 cap of mRNA by several metal

ion-macrocycle complexes.9 Recent collaborative work

between the Morrow and Richard laboratories has focused on

characterizing the rate acceleration for catalysis by metal ion

complexes and defining the origin of the catalytic rate

acceleration.Kinetic Analyses. An examination of the literature sug-

gested to us that the laboratory time expended in the synthe-

sis of novel metal ion catalysts of RNA cleavage oftenexceeded the time spent in characterizing their kinetics and

mechanism of action. In particular, it is difficult to compare the

catalytic activity of metal ion complexes prepared in differ-

ent laboratories, because there is no commonly agreed upon

protocol for measuring and reporting the kinetic parameters

for catalysis. Our initial goal was to develop such a protocol

for reporting kinetic data for metal ion complex-catalyzed

phosphate diester cleavage in water at 25 C, in the hope that

it might be adopted by other laboratories.

We have reported second-order rate constants kMe for the

catalyzed cleavage of HpPNP, UpPNP, and UpU to form cyclic

SCHEME 1

Phosphate Binding Energy and Catalysis Morrow et al.

540 ACCOUNTS OF CHEMICAL RESEARCH 539-548 April 2008 Vol. 41, No. 4


11/26

phosphate diesters. These rate constants are generally deter-

mined as the slopes of linear plots ofkobsd (s-1) for phosphate

diester cleavage against the catalyst concentration. The rate

constant kMe has the same units (M-1 s-1) and meaning as

kcat/Km for an enzyme-catalyzed reaction. Values ofkMe deter-

mined at Buffalo and elsewhere can be directly compared in

order to provide a simple and meaningful measure of the cat-

alytic activity of different metal ion complexes or of their activ-ity relative to enzyme catalysts.

The catalytic effectiveness of a variety of metal ion com-

plexes was then evaluated over a broad range of pH. Figure

1 shows representative pH-rate profiles for the Zn2(1)(H2O)-

and Zn(2)H2O-catalyzed cleavage of HpPNP and for the spon-

taneous specific-base-catalyzed cleavage of HpPNP.10 These

profiles show that the complex between HpPNP and the fully

protonated catalyst is inactive and that this complex is con-

verted to the active form upon loss of a proton. Maximal activ-

ity is observed at high pH, where the metal-bound water of

the free catalyst is ionized. The pH- and pD-rate profiles for

catalysis of the cleavage of HpPNP, UpPNP, and UpU by a

broad range of mono- and dinuclear catalysts are also gov-

erned by ionization of a metal-bound water, as shown in

Scheme 2.1019 The data in Figure 1 and that for related cat-

alyzed reactions show a good fit to eq 1 derived for Scheme

2, which gave values of Ka similar to those determined by

potentiometric titration.10

kMe ) [kZnKa

Ka + [H+]] (1)

Figure 1 shows that the relative activity of Zn2(1)(H2O) and

Zn(2)(H2O) as catalysts of the cleavage of HpPNP depends

upon whether the observed second-order rate constants kMeare compared at high or low pH. There is only a 12-fold dif-

ference in the apparentreactivity of the dinuclear complex and

the mononuclear complex at high pH. This difference

increases to 290-fold for reactions at low pH because thedownward break in catalytic activity is observed at a higher pH

for Zn(2)(H2O), as a result of the 1.2 unit higher pKa for ion-

ization of the zinc-bound water at Zn(2)(H2O) than of that at

Zn2(1)(H2O).10

The high catalytic activity of Zn2(1)(H2O) is notable because

tethering two mononuclear Zn2+ complexes to form a

dinuclear complex often causes only a statistical ca. 2-fold

increase in catalytic activity.15 The X-ray structure of crystals

(grown at pH 6.0) of the perchlorate salt of Zn2(1)(H2O) shows

that the two Zn2+ are separated by only 3.66 , due to shield-

ing of the electrostatic interaction between the metal cationsby the bridging alkoxide.10 The interactions between the two

Zn2+ and the linker alkoxide anion draw the metal cations

into a densely charged core, which provides a particularly

large electrostatic stabilization of the dianionic transition state

for phosphate diester cleavage.10,15

Transition State Binding Energies

Our next goal was to obtain a simple measure of catalytic

effectiveness from our kinetic data. The rate accelerations

for the catalyzed cleavage of HpPNP, given by eq 2 derived

for Scheme 3, were calculated from the relative displace-

ment of the parallel lines for the formally HO--catalyzed

cleavage ([, Figure 1) and the HO--dependent metal ion

complex catalyzed reactions (b and 9), where Ka is the ion-

ization constant for the metal-bound water and Kw ) 10-14

M2.20 The transition state binding energies ofGSq

) -9.6

and -5.9 kcal/mol for the reactions catalyzed by

Zn2(1)(H2O) (Ka ) 107.8 M) and Zn(2)(H2O) (Ka ) 10

9.2 M)

respectively, correspond to (1.1 107)-fold and (2.2

104)-fold rate accelerations over the specific-base-catalyzed

reaction.18,20 Zn2(1)(H2O) is an extremely impressive small

FIGURE 1. pH-rate profiles for cleavage of HpPNP catalyzed by

HO- and Zn(II) complexes in water at 25 C:10 ([) kobsd (s-1) for

spontaneous HO--catalyzed cleavage; (9), kMe (M-1 s-1) forcleavage catalyzed by Zn(2)(H2O); (b), kMe (M

-1 s-1) for cleavage

catalyzed by Zn2(1)(H2O); (1), ratio of kMe for cleavage catalyzed by

the dinuclear (kdi) and mononuclear (kmono) Zn(II) complexes.

SCHEME 2


Vol. 41, No. 4 April 2008 539-548 ACCOUNTS OF CHEMICAL RESEARCH 541


12/26

molecule catalyst. Metal ion complexes with even higher

catalytic activity in water21 and in methanol22 have been

prepared.

GSq

) GMeq

- GHOq

) -RTln[kZnKa/KwkHO ] (2)Figure 2 shows the transition state binding energy as the

difference in the activation barrier for HO--catalyzed cleav-

age of an RNA analogue and for the metal ion complex-cat-

alyzed reaction, which is also an HO--dependent reaction (eq

2). The calculation ofGSq compresses extensive kinetic data

into a single parameter that provides a direct measure for cat-

alytic activity at neutral pH, where the catalyst is largely pro-

tonated. We have determined the values ofGSq for a wide

variety of metal ion complex-catalyzed reactions and used

these data to evaluate the effect of changing catalyst and sub-

strate structure on catalytic activity.11,14,16,1820

Active Form of Catalyst

The pH dependence (Figure 1) of the uncatalyzed and cata-

lyzed cleavage of HpPNP at pH less than the catalyst pKashows that under these conditions a proton is lost from the

catalyst or substrate on proceeding to the rate-determining

transition state. This kinetic analysis cannot distinguish

between (a) the loss of proton from the substrate, in which

case Zn2(1)(H2O) is the active catalyst and (b) the loss of a pro-

ton from the catalyst, in which case the active form of the cat-

alyst is Zn2

(1)(HO-), whose concentration approaches a limit

at pH > pKa (Scheme 2).10,12

Analysis of inhibition of the Zn2(1)(H2O)-catalyzed cleav-

age of HpPNP shows that methylphosphate dianion binds to

Zn2(1)(H2O) with a 1600-fold higher affinity than does dieth-

ylphosphate monoanion.23 This strong and specific binding of

the stable dianion resembles the specific binding of the dian-

ionic transition state for cleavage of HpPNP to Zn2(1)(H2O),

with an affinity that is much greater than that of the substrate

monoanion.10We concluded that methyl phosphate dianion

is a transition state analogue for the Zn2(1)(H2O)-catalyzed

cleavage of phosphate diesters.24

Apparent inhibition constants, Ki, for inhibition of Zn2-

(1)(H2O)-catalyzed cleavage of HpPNP by methyl phosphate

dianion approach a limiting small value of 6 10-6 M for for-

mation of a tight complex at low pH, where the catalyst exists

mainly in the protonated form Zn2(1)(H2O).23 These data (not

shown) were fit to the model in Scheme 4 in which the tran-

sition state analogue dianion binds to the active protonated

catalyst, Zn2

(1)(H2

O), but not to the ionized catalyst

Zn2(1)(HO-). We concluded that Zn2(1)(H2O) is the active cat-

alyst that stabilizes the transition state dianion and that it is

converted to the inactive form Zn2(1)(HO-) when the pH is

increased above pKa ) 7.8. Deprotonation of Zn2(1)(H2O) may

inactivate the catalyst by changing the favorable displacement

of the water ligand by the substrate phosphate monoanion to

an unfavorable reaction for the displacement of the strongly

basic hydroxide ion ligand.

A change in solvent from H2O to D2O causes an increase

from 7.8 to 8.4 in the pKa of the zinc-bound water at

Zn2(1)(L2O) (Scheme 2), but has little effect on the reactivity of

SCHEME 3

FIGURE 2. Bar graph that shows the barrier for HO--promoted

phosphate diester cleavage in water (GHOq, green bar), the barrier

for the metal ion complex-catalyzed hydroxide ion-promoted

reaction (GMeq, green bar), and the binding energy for the

transition state for the aqueous HO--promoted reaction (GSq).

SCHEME 4




13/26

Zn2(1)(L2O) toward cleavage of UpPNP.12 Therefore, there is

no primary kinetic solvent deuterium isotope effect on the

cleavage reaction that results from movement of a hydron in

the rate-determining transition state,12 such as occurs in reac-

tions where there is concerted general base catalysis (GBC,

Scheme 5A). We concluded that the phosphate diester cleav-

age reaction follows the specific-base-catalyzed (SBC) path-

way shown in Scheme 5B.

We have proposed that the SBC pathway is observed for

catalysis by these metal ion complexes because of the dom-

inant role played by electrostatics in stabilization of the tran-

sition state for cleavage of RNA analogues by interactions withthe metal ion complex catalyst (Scheme 5B).12 In other words,

that the covalent-type stabilization gained by placing the

Brnsted base at the transition state for the GBC reaction

(Scheme 5A) is smaller than the electrostatic stabilization lost

upon partial neutralization of negative charge at the now

partly protonated O-2.

StructureReactivity Effects

We next used the above kinetic protocols to examine the

effect of systematic changes in the metal cation, ligand, and

substrate structure on the transition state binding energy GSq

(Scheme 3) for reactions catalyzed by free metal ions and by

metal ion complexes.

Ligand Effects. Chart 1 shows transition state binding

energies (GSq, Scheme 3) for catalysis of the cleavage of

HpPNP by several mononuclear Zn2+ complexes and by

hydrated Zn2+.18 By comparison, a value ofGSq

) -3.3

kcal/mol was determined for Zn2+-catalyzed deprotonation of

acetone,25 where Zn2+ interacts with a monoanionic rather

than a dianionic transition state. A value ofGSq

) -6.1 kcal/

mol was determined for Zn2+

-catalyzed aldose-ketose

isomerization of trioses, where there is additional stabiliza-

tion of the transition state from a chelate effect (Scheme 6).26

Zn2+ alone is a good catalyst of the cleavage of HpPNP

(Chart 1). However, it is not possible to obtain large rate

constants for this catalyzed cleavage at high pH, because of

the sparing solubility of hydrated Zn2+. Ligands 2-5

strongly enhance catalysis by increasing the total concen-

tration of soluble metal cation. The small variation in GSq

across Chart 1 suggests a similar origin for the transition

state stabilization, which we propose is mainly stabilizing

electrostatic interactions between the metal cation and tran-

sition state dianion.18

Chart 1 shows that macrocycle ligands either increase or

decrease the catalytic activity compared with the case where

there is no ligand but that the effect on GSq

is no more than1 kcal/mol. The formation of Zn2+ complexes has the effect

of shifting positive charge from Zn2+ to the electron-donor

ligand atom so that the weakest complexes will tend to show

the highest charge density at Zn2+. The complexes of Zn2+ to

macrocycles 4 and 5 are substantially weaker than the com-

plexes to 2 and 3, but the weakly bound Zn2+ at the former

complexes shows the greater catalytic activity (Chart 1).18 The

higher activity of Zn(4)(H2O) and Zn(5)(H2O) may be due to the

larger positive charge density at the more weakly coordinated

Zn2+, and the resulting enhancement of stabilization from

electrostatic interactions with the dianionic transition state.18

Cation Effects. The second-order rate constants for

cleavage of HpPNP catalyzed by Cu2(1)(H2O) (Chart 2) are

much smaller than those for catalysis by Zn2(1)(H2O) under

the same conditions and are invariant between pH 7 and

10.14 The X-ray crystal structure for Cu2(1)(H2O) shows a

bridging alkoxide linker and a Cu(II)-Cu(II) distance of 3.58

,27 similar to that observed in the crystal structure for

Zn2(1)(H2O).10 The structure for Zn2(1)(H2O) shows one

hexacoordinated and one pentacoordinated Zn(II),10 while

the structure for Cu2(1)(H2O) shows two pentacoordinated

SCHEME 5 CHART 1

SCHEME 6




14/26

Cu(II).27 We proposed that the coordination site missing

from Cu2(1)(H2O) is essential for the efficient binding and

catalysis of the reaction of HpPNP.

The larger absolute transition state binding energy ofGSq

) 9.8 kcal/mol for cleavage of HpPNP catalyzed by Eu(III)

(mainly [Eu(OH2)9]3+)19 in water than for cleavage catalyzed

by hydrated Zn2+ in water (GSq

) 6.6 kcal/mol, Chart 1)18 is

probably due to the stronger stabilizing electrostatic interac-

tion of a trication than of a dication with the transition state

dianion. Chart 3 gives the values ofGSq for catalysis of cleav-

age of HpPNP by mononuclear Eu(III) complexes of 616 and7,19 and for a dinuclear complex that forms by spontaneous

dimerization of [Eu(6)(OH2)2]+ at pH g 7.5.16

The absolute value ofGSq

) 8.6 kcal/mol for catalysis by

[Eu(7)(OH2)2]3+ is only 1.2 kcal/mol larger than that observed

for the most active mononuclear Zn(II) catalysts (Chart 1).

Apparently, the larger number of donor groups to Eu(III) at

[Eu(7)(OH2)2]3+ compared with mononuclear Zn(II) catalysts act

to attenuate the 3.2 kcal/mol larger transition state stabiliza-

tion that is observed for catalysis by Eu(III) when all the ligands

are water. The 1.5 kcal/mol larger absolute value ofGSq for

[Eu(7)(OH2)2]3+ than for [Eu(6)(OH2)2]

+ shows that there is a

small increase in the catalytic efficiency with increasing total

positive charge at the catalyst available to interact with the

transition state dianion. Finally, the similar transition state

binding energies for catalysis by [Eu(6)(OH2)2]+ and [Eu2-

(6)2(OH)(OH2)2]+

provide evidence that the two metal ions in

the dimeric complex operate independently in catalyzing the

cleavage of HpPNP.

Specificity. There are significant differences in the rate

accelerations for the Zn2(1)(H2O)-catalyzed cleavage of RNA

analogues and a dinucleoside monophosphate that reflect the

different specificities of the dinuclear complex Zn2(1)(H2O) fortransition state binding (Chart 4).10,11,17,20 The binding site

at this catalyst has not been characterized; however, it should

not show strong shape complementarity to any of these sub-

strates. We proposed other origins for the range of transition

state binding energies shown in Chart 4.

(1)The difference between GSq

) -9.6 and -7.2 kcal/mol

for catalysis of cleavage of HpPNP, a minimal substrate,

and of the more bulky substrate UpPNP, respectively, sug-

gests that close approach of the phosphate diester to the

metal cations is required for effective catalysis and that

there is steric hindrance to the approach of the larger sub-

strate UpPNP to Zn2(1)(H2O).11



for catalysis of cleavage of UpOEt and UpOCH2CCl3, respec-

tively, is a consequence of the different Brnsted parame-

ters lg ) -1.28 and -0.72 for the specific-base-catalyzed

and Zn2(1)(H2O)-catalyzed cleavage reactions.17,28 This cor-

responds to the neutralization of an effective transition

state charge of ca. 0.56 units by interactions between the

catalyst and the leaving group anion.29 These data show

that there is stabilization of the transition state for cleav-age of UpOEt from interaction of the cationic catalyst with

both the reacting phosphate core and the strongly basic

alkoxy leaving group.



for catalysis of cleavage of UpU and UpPNP, respectively,

is due partly or entirely to transition state stabilization by

interaction of the catalyst with the strongly basic alkoxy

leaving group at UpU. There may also be a weak nonspe-

cific interaction between the catalyst and the second pyri-

midine base at UpU.20

CHART 2

CHART 3

CHART 4




15/26

Lessons from Studies on Small MoleculeCatalysis

The high catalytic activity of Zn2(1)(H2O) arises from strong

stabilizing binding interactions (GSq, Scheme 3) between the

dianionic transition state and the densely charged cationic

core of Zn2(1)(H2O).10 No concerted general base catalysis ofphosphate diester cleavage is observed, presumably because

anything gained from such catalysis is offset by an even larger

reduction in the electrostatic stabilization of the transition state

(Scheme 5).

A distinguishing feature of these metal ion complex-cata-

lyzed reactions is their large discrimination between the bind-

ing of the substrate phosphate diester monoanion (weak) and

of the oxyphosphorane-like transition state dianion30 and the

methyl phosphate dianion transition state analogue (strong).23

These results provide evidence that transition state stabiliza-tion by mononuclear and dinuclear metal ion complexes is

due largely to electrostatic interactions, which are optimal for

the dianionic transition state and transition state analogue. In

other words, good catalysis of the cleavage of phosphate

diesters in water has been obtained relatively easily because

of the intrinsically strong stabilizing interactions between

densely charged metals and phosphate dianions. The total

transition state binding energy GSq can be modified by inter-

actions between the metal ion and its ligands, but these effects

are small in comparison with the transition state stabilization

observed for catalysis by free metal ions, where the ligandsare water (Chart 1).

Enzyme Catalysis

A major difference between small molecule and enzyme cat-

alysts is that the latter have evolved mechanisms for utiliza-

tion of the binding interactions between the protein and

nonreacting portions of the substrate in stabilization of the

transition state for the catalyzed reaction.2 The transition state

binding energies of up to 10 kcal/mol observed for cataly-

sis of the cleavage of phosphate diesters are due mainly toelectrostatic interactions between the catalyst and the phos-

phate group, which is the reaction center. We have observed

even larger transition state stabilizations of ca. 12 kcal/mol

from utilization of the binding energy between enzyme cata-

lysts and the nonreacting phosphodianion group of substrates

for enzyme-catalyzed proton transfer, hydride transfer, and

decarboxylation reactions.3133

Reactions of Triosephosphates

Triosephosphate isomerase (TIM) is a prototypical catalyst of

proton transfer at carbon (Chart 5). This enzyme catalyzes the

stereospecific 1,2-hydrogen shift at (R)-glyceraldehyde 3-phos-

phate (GAP) to give dihydroxyacetone phosphate (DHAP). A

single base (Glu-165) catalyzes suprafacial proton transfer

through a cis-enediol(ate) intermediate.34 The ratio of second-

order rate constants (kcat/Km)GAP/(kcat/Km)GA ) (2.4 108 M-1

s-1)/(0.34 M-1 s-1) ) 7 108 for the TIM-catalyzed isomer-ization of GAP and of (R)-glyceraldehyde shows that binding

interactions between the enzyme and the phosphodianion

group of GAP provide g12 kcal/mol of transition state stabi-

lization.35 This may account for all but 2 kcal/mol of the tran-

sition state stabilization for TIM-catalyzed isomerization of

GAP.35,36

The value of (kcat/Km)Gly ) 0.26 M-1 s-1 for TIM-catalyzed

exchange of deuterium from solvent D2O into the truncated

substrate glycolaldehyde is similar to (kcat/Km)GA ) 0.34 M-1

s-1 for isomerization of (R)-glyceraldehyde.31 The deuterium

exchange reaction of glycolaldehyde is strongly activated by

addition of the second substrate piece phosphite dianion,

HPO32-. The data give (kcat/Km)EHPi ) 185 M

-1 s-1 for turn-

over of glycolaldehyde by TIM that is saturated with phos-

phite dianion, so binding of phosphite dianion to TIM results

in a 700-fold rate acceleration of proton transfer from car-

bon. The two substrate pieces, glycolaldehyde and HPO32-,

each bind weakly to TIM, and there is a large chelate effect

for binding of the whole substrate GAP (Chart 5).31,37 The

binding of HPO32- to free TIM (Kd ) 38 mM) is 700-fold

weaker than its binding to the fleeting TIM transition state

complex (Kdq

) 53 M, Scheme 7). This corresponds to an

intrinsic phosphite dianion binding energy of -5.8 kcal/mol

(Scheme 7).31

The proton transfer reaction catalyzed by TIM occurs in an

active site at which the substrate is sequestered from

solvent.38,39 We proposed a model for catalysis by TIM in

CHART 5




16/26

which part of the total intrinsic binding energy of the phos-

phodianion group of substrate is utilized to drive a protein

conformational change that sequesters the substrate at an

active site with an apparent dielectric constant substantiallylower than that of solvent and with the catalytic groups opti-

mally organized to stabilize the transition state for deproto-

nation of R-carbonyl carbon.31,40

The whole substrate DHAP is reduced by NADH to give L-glyc-

erol 3-phosphate in a reaction catalyzed by R-glycerol phosphatedehydrogenase (Scheme 8).41 The ratio (kcat/Km)DHAP/(kcat/Km)Gly) (1 106 M-1 s-1)/(0.009 M-1 s-1) ) 1.1 108 for rabbit

muscle R-glycerol phosphate dehydrogenase-catalyzed reduc-

tion of DHAP and glycolaldehyde gives an intrinsic phosphate

binding energy of -11 kcal/mol, which is similar to that for

TIM.33We find that phosphite dianion is also a powerful activa-

tor of enzyme-catalyzed reduction of the truncated neutral sub-

strate glycolaldehyde by NADH.33 X-ray crystallographic analysis

of human R-glycerol phosphate dehydrogenase provides evi-

dence that the phosphate binding energy of the substrate is used

to drive the closure of a loop over the substrate.41

Orotidine 5-MonophosphateDecarboxylase (OMPDC)

OMPDC is a remarkable enzyme that effects an enormous

1017-fold acceleration of the chemically very difficult decar-

boxylation of orotidine 5-monophosphate (OMP, Chart 6)

to give uridine 5-monophosphate (UMP).42 The enzymat-

ic43 and nonenzymatic44 decarboxylation reactions pro-

ceed through the vinyl carbanion intermediates.

Comparison of the X-ray crystal structures of free yeast

OMPDC and that complexed with the transition state ana-

logue 6-hydroxyuridine 5-monophosphate shows that

ligand binding results in a large motion to close the active

site with the formation of numerous proteinligand con-

tacts, including five hydrogen bonds to the phosphodian-

ion group.45 The phosphodianion at OMP, or at anyphosphorylated enzymatic substrate, may simply anchor

the substrate to the enzyme, or the enzyme conformational

change effected by the small remote group may directly

assist in the creation of an active site that provides opti-

mal transition state stabilization.

The intrinsic binding energy of the phosphodianion group

of OMP, calculated from the ratio of second-order rate con-

stants kcat/Km for the OMPDC-catalyzed reactions of OMP (9.4

106 M-1 s-1)46 and the truncated substrate 1-(-D-erythro-

furanosyl)orotic acid (EO, 2.1 10-2 M-1 s-1)32 is -12 kcal/

mol (Chart 6). The weak binding of the EO (Kd 0.1 M) and

phosphite dianion (Kd

) 0.14 M) pieces to OMPDC is striking

in view of the tight binding of the whole substrate OMP (Km) 1.6 M).37,46 Decarboxylation of EO is strongly activated by

phosphite dianion, with (kcat/Km)EHPi ) 1600 M-1 s-1 for turn-

over of EO by OMPDC that is saturated with HPO32-, so the

binding of this dianion to OMPDC results in an 80000-fold

acceleration of decarboxylation.32 The binding of HPO32- to

OMPDC (Kd ) 0.14 M) must also be 80000-fold weaker than

its binding to the fleeting complex of OMPDC with EO in the

transition state (Kdq

) 1.8 M, Scheme 7). The total intrinsic

binding energy of HPO32- at the transition state complex is

therefore -7.8 kcal/mol. This is divided into the small -1.2

SCHEME 7

SCHEME 8

CHART 6




17/26

kcal/mol binding energy observed in the ground-state com-

plex and an additional -6.6 kcal/mol interaction that devel-

ops on proceeding to the transition state for enzyme-catalyzed

decarboxylation of EO.2,32

The Pauling ParadigmOur studies have shown that the interactions between non-

reacting phosphodianion fragments of substrate and the

protein provide an 1112 kcal/mol stabilization of the tran-

sition states for proton transfer, hydride transfer, and decar-

boxylation reactions. Therefore, enzymes that catalyze the

reactions of small phosphorylated substrates such as GAP

and DHAP have a potential transition state binding energy

of -12 kcal/mol. The medium-sized phosphorylated sub-

strate OMP interacts with OMPDC via its phosphodianion,

ribose sugar ring, and nucleic acid base portions and exhib-

its a transition state binding energy far in excess of -15

kcal/mol that was proposed as the limit on noncovalent

enzyme catalysis.3,42

Pyridoxal 5-phosphate (PLP) provides impressive covalent

catalysis of the deprotonation ofR-amino acids to form R-amino

carbanions.47 However, the large rate acceleration for proton

transfer obtained by recruitment of this cofactor can be also

obtained by protein catalysts, such as proline racemase. This

enzyme provides a 19 kcal/mol stabilization of the transition

state for deprotonation of enzyme-bound proline by interaction

with the protein catalyst.48

Our proposal that pure protein catal-ysis of deprotonation of amino acids results from the binding of

the amino acid substrate at a nonpolar active site that favors for-

mation of a carbanion zwitterion49,50 has been supported by

X-ray crystallographic and computational studies.51,52 We are

generally skeptical of arguments that cofactors such as PLP have

evolved to do what is impossible for protein catalysts, because of

the great diversity of protein-catalyzed reactions. On the other

hand, cofactor catalysis is often simpler and more general than

protein catalysis, and these factors favor the recruitment of cofac-

tors in enzymatic catalysis.

We acknowledge the NSF (Grant CHE0415356) and the NIH

(Grant GM39754) for support of our work, and we thank our

co-workers named in the references for their many contribu-

tions to our research.

BIOGRAPHICAL INFORMATION

Janet R. Morrow received a B.S. degree in chemistry from theUniversity of California, Santa Barbara, and her Ph.D. degree inchemistry (1985) from the University of North Carolina, ChapelHill. She is currently Professor of Chemistry at the University at

Buffalo, SUNY.

Tina L. Amyes received B.A. and M.A. degrees in natural sci-ences and her Ph.D. degree in chemistry (1986) from the Univer-sity of Cambridge (England). She is currently an Adjunct AssociateProfessor at the University at Buffalo, SUNY.

John P. Richard received a B.S. degree in biochemistry and hisPh.D. degree in chemistry (1979) from The Ohio State Univer-sity. He is currently Professor of Chemistry at the University at Buf-falo, SUNY.

FOOTNOTES

*E-mail: [email protected].

REFERENCES

1 Pauling, L. The nature of forces between large molecules of biological interest.Nature1948, 161, 707709.

2 Jencks, W. P. Binding energy, specificity and enzymic catalysis: The circe effect.Adv. Enzymol. Relat. Areas Mol. Biol. 1975, 43, 219410.

3 Zhang, X.; Houk, K. N. Why enzymes are proficient catalysts: Beyond the Paulingparadigm. Acc. Chem. Res. 2005, 38, 379385.

4 Morrow, J.; Iranzo, O. Synthetic metallonucleases for RNA cleavage. Curr. Opin.Chem. Biol. 2004, 8, 192200.

5 Williams, N. H.; Takashi, B.; Wall, M.; Chin, J. Structure and nuclease activity ofsimple dinuclear metal complexes: quantitative dissection of the role of metal ions.Acc. Chem. Res. 1999, 32, 485493.

6 Blasko, A.; Bruice, T. C. Recent studies of nucleophilic, general-acid and metal ioncatalysis of phosphate diester hydrolysis. Acc. Chem. Res. 1999, 32, 475484.

7 Chin, K. O. A.; Morrow, J. R. RNA cleavage and phosphate diester transesterificationby encapsulated lanthanide ions: Traversing the lanthanide series withlanthanum(III), europium(III), and lutetium(III) complexes of 1,4,7,10-tetrakis(2-hydroxyalkyl)-1,4,7,10-tetraazacyclododecane. Inorg. Chem. 1994, 33,50365041.

8 Morrow, J. R.; Buttrey, L. A.; Shelton, V. M.; Berback, K. A. Efficient catalyticcleavage of RNA by lanthanide(III) macrocyclic complexes: Toward syntheticnucleases for in vivo applications. J. Am. Chem. Soc. 1992, 114, 19031905.

9 Baker, B. F.; Khalili, H.; Wei, N.; Morrow, J. R. Cleavage of the 5 cap structure ofmRNA by a europium(III) macrocyclic complex with pendant alcohol group. J. Am.

Chem. Soc. 1997, 119, 87498755.10 Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. Physical and kinetic

analysis of the cooperative role of metal ions in catalysis of phosphodiester cleavageby a dinuclear Zn(II) complex. J. Am. Chem. Soc. 2003, 125, 19881993.

11 Yang, M.-Y.; Richard, J. P.; Morrow, J. R. Substrate specificity for catalysis ofphosphodiester cleavage by a dinuclear Zn(II) complex. Chem. Commun. 2003,28322833.

12 Yang, M.-Y.; Iranzo, O.; Richard, J. P.; Morrow, J. R. Solvent deuterium isotopeeffects on phosphodiester cleavage catalyzed by an extraordinarily active Zn(II)complex. J. Am. Chem. Soc. 2005, 127, 10641065.

13 Feng, G.; Mareque-Rivas, J. C.; Williams, N. H. Comparing a mononuclear Zn(II)complex with hydrogen bond donors with a dinuclear Zn(II) complex for catalysingphosphate ester cleavage. Chem. Commun. 2006, 18451847.

14 Iranzo, O.; Richard, J. P.; Morrow, J. R. Structure -activity studies on the cleavageof an RNA analogue by a potent dinuclear metal ion catalyst. The effect of changingthe metal ion. Inorg. Chem. 2004, 43, 17431750.

15 Iranzo, O.; Elmer, T.; Richard, J. P.; Morrow, J. R. Cooperativity between metal ionsin the cleavage of phosphate diesters and RNA by dinuclear Zn(II) catalysts. Inorg.Chem. 2003, 42, 77377746.

16 Farquhar, E.; Richard, J. P.; Morrow, J. R. Formation and stability of mononuclearand dinuclear Eu(III) complexes and their catalytic reactivity toward cleavage of anRNA analogue. Inorg. Chem. 2007, 46, 71697177.

17 Yang, M.-Y. Ph.D. Thesis, University at Buffalo, 2007.

18 Mathews, R. A.; Rossiter, C. S.; Morrow, J. R.; Richard, J. P. A minimalist approachto understanding the efficiency of mononuclear Zn(II) complexes as catalysts ofcleavage of an RNA analogue. Dalton Trans. 2007, 38043811.

19 Nwe, K.; Richard, J. P.; Morrow, J. R. Direct excitation luminescence spectroscopyof Eu(III) complexes of 1,4,7-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecanederivatives and kinetic studies of their catalytic cleavage of an RNA analog. DaltonTrans. 2007, 51715178.

20 ODonoghue, A.; Pyun, S. Y.; Yang, M.-Y.; Morrow, J. R.; Richard, J. P. Substratespecificity of an active dinuclear complex for cleavage of RNA analogues and a

dinucleoside. J. Am. Chem. Soc. 2006, 128, 16151621.




18/26

21 Feng, G.; Mareque-Rivas, J. C.; Torres Martin de Rossales, R.; Williams, N. H. Ahighly reactive mononuclear Zn(II) complex for phosphodiester cleavage. J. Am.Chem. Soc. 2005, 127, 1347013471.

22 Neverov, A. A.; Lu, Z. L.; Maxwell, C. I.; Mohamed, M. F.; White, C. J.; Tsang,J. S. W.; Brown, R. S. Combination of a dinuclear Zn 2+ complex and a mediumeffect exerts a 1012-fold rate enhancement of cleavage of an RNA and DNA modelsystem. J. Am. Chem. Soc. 2006, 128, 1639816405.

23 Yang, M.-Y.; Morrow, J. R.; Richard, J. P. A transition state analog for phosphate

diester cleavage catalyzed by a small enzyme-like metal ion complex. Bioorg. Chem.2007, 35, 366374.

24 Wolfenden, R. Transition state analogues for enzyme catalysis. Nature1969, 223,704705.

25 Crugeiras, J.; Richard, J. P. A comparison of the electrophilic reactivities of Zn 2+

and acetic acid as catalysts of enolization: imperatives for enzymatic catalysis ofproton transfer at carbon. J. Am. Chem. Soc. 2004, 126, 51645173.

26 Nagorski, R. W.; Richard, J. P. Mechanistic imperatives for aldose-ketoseisomerization in water: specific-, general base- and metal ion-catalyzedisomerization of glyceraldehyde with proton and hydride transfer. J. Am. Chem. Soc.2001, 123, 794802.

27 Fry, F. H.; Moubaraki, B.; Murray, K. S.; Spiccia, L.; Warren, M.; Skelton, B. W.;White, A. H. Asymmetry in endogenously bridged binuclear copper(II) and zinc(II)complexes formed by 1,2-bis[1,4,7-triazacyclonon-1-yl]-propan-2-ol. Dalton Trans.2003, 866871.

28 Kosonen, M.; Youseti-Salakdeh, E.; Stromberg, R.; Lnnberg, H. Mutual

isomerization of uridine 2- and 3

-alkylphosphates and cleavage to a 2

,3

-cyclicphosphate: the effect of the alkyl group on the hydronium- and hydroxide-ion-

catalyzed reactions. J. Chem. Soc., Perkin Trans. 21997, 26612666.29 Williams, A. Electronic charge and transition state structure in solution. Adv. Phys.

Org. Chem. 1992, 27, 155.30 Perreault, D. M.; Anslyn, E. V. Unifying the current data on the mechanism of

cleavage-transesterification of RNA. Angew. Chem., Int. Ed. Engl. 1997, 36, 432450.

31 Amyes, T. L.; Richard, J. P. Enzymatic catalysis of proton transfer at carbon:Activation of triosephosphate isomerase by phosphite dianion. Biochemistry2007,46, 58415854.

32 Amyes, T. L.; Richard, J. P.; Tait, J. J. Activation of orotidine 5-monophosphatedecarboxylase by phosphite dianion: the whole substrate is the sum of two parts.J. Am. Chem. Soc. 2005, 127, 1570815709.

33 Tsang, W.-Y.; Amyes, T. L.; Richard, J. P. A Substrate in Pieces: Allosteric Activationof Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosph ite Dianon, Biochemistry,submitted for publication.

34 Knowles, J. R.; Albery, W. J. Perfection in enzyme catalysis: The energetics of

triosephosphate isomerase. Acc. Chem. Res. 1977, 10, 105111.35 Amyes, T. L.; ODonoghue, A. C.; Richard, J. P. Contribution of phosphate intrinsic

binding energy to the enzymatic rate acceleration for triosephosphate isomerase.J. Am. Chem. Soc. 2001, 123, 1132511326.

36 Richard, J. P. Acid-base catalysis of the elimination and isomerization reactions oftriose phosphates. J. Am. Chem. Soc. 1984, 106, 49264936.

37 Jencks, W. P. On the attribution and additivity of binding energies. Proc. Natl. Acad.Sci. U.S.A. 1981, 78, 40464050.

38 ODonoghue, A. C.; Amyes, T. L.; Richard, J. P. Hydron transfer catalyzed bytriosephosphate isomerase. Products of isomerization of (R)-glyceraldehyde 3-phosphate in D2O. Biochemistry2005, 44, 26102621.

39 ODonoghue, A. C.; Amyes, T. L.; Richard, J. P. Hydron transfer catalyzed bytriosephosphate isomerase. Products of isomerization of dihydroxyacetonephosphate in D

2O. Biochemistry2005, 44, 26222631.

40 Sampson, N. S.; Knowles, J. R. Segmental motion in catalysis: Investigation of ahydrogen bond critical for loop closure in the reaction of triosephosphate isomerase.Biochemistry1992, 31, 84888494.

41 Ou, X.; Ji, C.; Han, X.; Zhao, X.; Li, X.; Mao, Y.; Wong, L.-L.; Bartlam, M.; Rao, Z.Crystal structure of human glycerol 3-phosphate dehydrogenase (GPDI). J. Mol. Biol.2006, 357, 858869.

42 Radzicka, A.; Wolfenden, R. A proficient enzyme. Science1995, 267, 9093.

43 Toth, K.; Amyes, T. L.; Wood, B. M.; Chan, K.; Gerlt, J. A.; Richard, J. P. Productdeuterium isotope effect for orotidine 5-monophosphate decarboxylase: Evidencefor the existence of a short-lived carbanion intermediate. J. Am. Chem. Soc. 2007,129, 1294612947.

44 Sievers, A.; Wolfenden, R. Equilibrium of formation of the 6-carbanion of UMP, apotential intermediate in the action of OMP decarboxylase. J. Am. Chem. Soc. 2002,124, 1398613987.

45 Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M. V.; Short, S. A. Anatomy ofa proficient enzyme: The structure of orotidine 5 -monophosphate decarboxylase inthe presence and absence of a potential transition state analog. Proc. Natl. Acad.Sci. U.S.A. 2000, 97, 20112016.

46 Porter, D. J. T.; Short, S. A. Yeast orotidine-5 -phosphate decarboxylase: Steady-state and pre-steady-state analysis of the kinetic mechanism of substratedecarboxylation. Biochemistry2000, 39, 1178811800.

47 Toth, K.; Richard, J. P. Covalent catalysis by pyridoxal: Evaluation of the effect of thecofactor on the carbon acidity of glycine. J. Am. Chem. Soc. 2007, 129, 30133021.

48 Williams, G.; Maziarz, E. P.; Amyes, T. L.; Wood, T. D.; Richard, J. P. Formation andstability of the enolates of N-protonated proline methyl ester and proline zwitterion inaqueous solution: a nonenzymatic model for the first step in the racemization ofproline catalyzed by proline racemase. Biochemistry2003, 42, 83548361.

49 Rios, A.; Amyes, T. L.; Richard, J. P. Formation and stability of organic zwitterions inaqueous solution: Enolates of the amino acid glycine and its derivatives. J. Am.Chem. Soc. 2000, 122, 93739385.

50 Richard, J. P.; Amyes, T. L. On the importance of being zwitterionic: Enzymiccatalysis of decarboxylation and deprotonation of cationic carbon. Bioorg. Chem.2004, 32, 354366.

51 Puig, E.; Garcia-Viloca, M.; Gonzalez-Lafont, A.; Lluch, J. M. On the ionization stateof the substrate in the active site of glutamate racemase. A QM/MM study about theimportance of being zwitterionic. J. Phys. Chem. A 2006, 110, 717725.

52 Pillai, B.; Cherney, M. M.; Diaper, C. M.; Sutherland, A.; Blanchard, J. S.; Vereras,J. C.; James, M. N. G. Structural insights into stereochemical inversion bydiaminopimilate epimerase: an antibacterial drug target. Proc. Natl. Acad. Sci.U.S.A. 2006, 103, 86688673.




19/26

Accelerated Publications

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase(NAD+) by Phosphite Dianion

Wing-Yin Tsang, Tina L. Amyes, and John P. Richard*Department of Chemistry, UniVersity at Buffalo, State UniVersity of New York, Buffalo, New York 14260-3000

ReceiVed January 30, 2008; ReVised Manuscript ReceiVed March 5, 2008

ABSTRACT: The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate(DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydro-genase (NAD+, GPDH) saturated with NADH is (1.0 106 M-1 s-1)/(8.7 10-3 M-1 s-1) ) 1.1 108,which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphitedianion binds very weakly to GPDH (Kd > 0.1 M), but the bound dianion strongly activates GLY towardenzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressedonly at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M-2 s-1)/(8.7

10

-3

M

-1

s

-1

))

5

10

5

M

-1

, which was used to calculate an intrinsic phosphite binding energy of-

7.7kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzedreduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structuraldata provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at amodular active site featuring (1) a binding pocket complementary to the reactive substrate fragment whichcontains all the active site residues needed to catalyze the reaction of the substrate piece or of the wholesubstrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement offlexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associatedprotein conformational changes that accompany the binding of phosphite dianion and/or phosphodianionsubstrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placementof the active site residues in positions where they provide optimal stabilization of the transition state forthe catalyzed reaction.

Enzymes are protein catalysts that effect rate accelerationsmuch larger than those observed for small molecule catalysts.The larger rate accelerations for enzymes are a consequenceof the greater binding affinities of enzymes for their reactiontransition states (1). Enzyme catalysis is so efficient that therelease of products would be intolerably slow if they were

to bind with the same affinity as the transition state. Forexample, the ca. 30 kcal/mol transition state intrinsic bindingenergy estimated for the enzymatic decarboxylation oforotidine 5-monophosphate (OMP)1 catalyzed by orotidine

This work was supported by Grant GM39754 from the NationalInstitutes of Health.

* To whom correspondence should be addressed. Telephone: (716)645-6800, ext. 2194. Fax: (716) 645-6963. E-mail: [email protected].

1 Abbreviations: OMP, orotidine 5-monophosphate; OMPDC, orotidine5-monophosphate decarboxylase; GAP, (R)-glyceraldehyde 3-phosphate;TIM, triosephosphate isomerase; GPDH, glycerol 3-phosphate dehydro-genase (NAD+); DHAP, dihydroxyacetone phosphate; NADH, nicotina-mide adenine dinucleotide, reduced form; R-GP, L-glycerol 3-phosphate;GLY, glycolaldehyde; BSA, bovine serum albumin; DTT, D,L-dithiothrei-tol; TEA, triethanolamine; NMR, nuclear magnetic resonance.

Copyright 2008 by the American Chemical Society Volume 47, Number 16 April 22, 2008

10.1021/bi8001743 CCC: $40.75 2008 American Chemical SocietyPublished on Web 04/01/2008


20/26

5-monophosphate decarboxylase (OMPDC) (2) is evenlarger than the 20 kcal/mol binding energy associated withthe effectively irreversible binding of biotin to avidin (3).Consequently, to avoid this free energy trap, enzymesgenerally bind their substrates and/or products much moreweakly than the reaction transition state.

It has been proposed that enzymes often produce largerate accelerations through the utilization of binding interac-tions between the protein and nonreacting portions of the

substrate for transition state stabilization (4, 5). However,the mechanism(s) by which enzyme catalysts selectivelyturn on these binding interactions at the transition statefor their catalyzed reactions is not generally known, and thisquestion is often omitted from discussions of the origin ofthe rate acceleration for enzyme catalysts.

The binding energy of the remote nonreacting phosphodi-anion groups of OMP and (R)-glyceraldehyde 3-phosphate(GAP) has been shown to provide a ca. 12 kcal/molstabilization of the transition states for OMPDC-catalyzeddecarboxylation (6) and proton transfer catalyzed by triose-phosphate isomerase (TIM) (7), respectively. Moreover, thesmall substrate piece phosphite dianion provides strongallosteric-like activation of OMPDC-catalyzed decarboxy-lation (6) and TIM-catalyzed deprotonation (8) of a secondtruncated substrate piece from which the phosphodianiongroup has been excised. The latter results show that asubstantial fraction of the intrinsic phosphate bindingenergy is expressed as transition state stabilization even inthe absence of a covalent connection between the substratepieces. Phosphite dianion binds weakly to both TIM (Kd )38 mM) (8) and OMPDC (Kd ) 140 mM) (6) so that thelarge intrinsic phosphite binding energy is expressed onlyat the respective transition states for these enzyme-catalyzedreactions.

X-ray crystallographic analyses suggest that both TIM andOMPDC utilize the binding energy of the substrate phos-phodianion group to drive the thermodynamically unfavor-able closure of a mobile protein loop(s) to cover the boundphosphodianion (912). We have proposed (8) that this loopclosure sequesters the substrate from bulk solvent and resultsin formation of a local microenvironment in which there isoptimal stabilization of the transition state for the enzymaticreaction (13, 14).

We examine here whether the functional equivalence ofGAP and the two-part substrate [glycolaldehyde + phos-phite] (Scheme 1) for proton transfer catalyzed by TIM mightalso be observed for other types of enzymatic reactions.

Glycerol 3-phosphate dehydrogenase (NAD+

-linked, EC1.1.1.8, GPDH) is used in the assay for TIM-catalyzedisomerization of GAP to couple formation of the product

dihydroxyacetone phosphate (DHAP) to its reduction byNADH to give L-glycerol 3-phosphate [R-GP (Scheme 2)](15). We report here that GPDH also catalyzes the very slowreduction of the neutral minimal substrate glycolaldehyde(GLY) by NADH and that the separate binding of the secondsubstrate piece phosphite dianion strongly activates GPDHfor catalysis of the reduction of GLY by NADH. Weconclude that the substrate pieces [glycolaldehyde + phos-phite] are able to effectively substitute for the whole substrateGAP or DHAP in enzyme-catalyzed proton transfer andhydride transfer reactions, respectively.

MATERIALS AND METHODS

Materials. Glycerol 3-phosphate dehydrogenase (lyophi-lized powder, 75 units/mg) from rabbit muscle was purchasedfrom USB Corp. Bovine serum albumin, fraction V (BSA),was from Roche. Dihydroxyacetone phosphate (lithium salt),NADH (disodium salt), glycolaldehyde dimer, triethanola-mine hydrochloride, D,L-dithiothreitol (DTT), and sodiumdeuteroxide (40 wt %, 99.9% D) were from Sigma-Aldrich.Deuterium oxide (99.9% D) was from Cambridge IsotopeLaboratories. Sodium phosphate (dibasic) was from Fluka,and sodium phosphite (dibasic, pentahydrate) was fromRiedel-de Haen (Fluka). Water was from a Milli-Q Academicpurification system. All other chemicals were reagent gradeor better and were used without further purification.

Preparation of Solutions.The solution pH was determinedas described previously (8). Stock solutions of NADH were

prepared by dissolving NADH (disodium salt) in water andwere stored at 4 C. The concentration of NADH wasdetermined spectrophotometrically at 340 nm using an of6220 M-1 cm-1. Stock solutions of DHAP were preparedon the day of the experiment by dissolving the lithium saltin water and adjusting the pH to 7.5 using 1 M NaOH andwere stored on ice. The concentration of DHAP wasdetermined spectrophotometrically at 340 nm as the con-centration of NADH oxidized in a reaction catalyzed byGPDH. GPDH (2 or 20 mg/mL) in 20 mM triethanolamine(TEA) buffer (pH 7.5, 32% free base) was dialyzed

exhaustively against the same buffer at 5

C. The concentra-tion of GPDH was determined from the absorbance at 280nm using an A of 5.15 for a 1% solution of GPDH (16) anda subunit molecular mass of 37500 Da. Stock solutions ofglycolaldehyde (100 mM monomer) were prepared bydissolving glycolaldehyde dimer in water and storing thesolution for at least 3 days at room temperature to allow forbreakdown of the dimer to the monomer (8). An apparentpKa of 6.38 for phosphite monoanion at 25 C and an I of0.12 (NaCl) was determined as the pH of a 30 mM solutionof the sodium salt at a 1:1 monoanion:dianion ratio.

Enzyme Assays. Dilutions of the dialyzed stock solutionsof 2 mg/mL enzyme (used for assays of GPDH-catalyzed

reduction of DHAP) and 20 mg/mL enzyme (used for assaysof GPDH-catalyzed reduction of GLY) were made in 20 mMTEA (pH 7.5, 32% free base) containing 10 mM DTT.

Scheme 1 Scheme 2

4576 Biochemistry, Vol. 47, No. 16, 2008 Accelerated Publication


21/26

All enzyme assays were conducted at 25 C and an I of0.12 (NaCl) in a volume of 1 mL. Initial velocities ofoxidation of NADH (e5% reaction) were calculated fromthe changes in absorbance at 340 nm using a of 6220M-1 cm-1. The standard assay mixture for GPDH activitycontained 100 mM TEA (pH 7.5), 1.2 mM DHAP, 0.20 mMNADH, 0.1 mg/mL BSA, 50 M DTT, and ca. 1 nM GPDHat an I of 0.12 (NaCl). Reactions were initiated by making

a 200-fold dilution of the diluted stock enzyme containing10 mM DTT into the reaction mixture. The same procedurewas used to determine the velocity of GPDH-catalyzedreduction over a range of initial concentrations of DHAP,except that the buffer was 20 mM TEA (pH 7.5).

Assay mixtures for the GPDH-catalyzed reduction of GLYin the absence of phosphite contained 10 mM TEA (pH 7.5),20 mM GLY (total), 0.20 mM NADH, and up to ca. 0.2mM GPDH at an I of 0.12 (NaCl). Assay mixtures for theGPDH-catalyzed reduction of GLY in the presence ofphosphite contained 10 mM TEA (pH 7.5), 2-60 mM GLY(total), 0.20 mM NADH, up to 32 mM phosphite dianion,and 0.6 M GPDH at an I of 0.12 (NaCl).

1H NMR Analyses. 1H NMR spectra at 500 MHz wererecorded in D2O at 25 C using a Varian Unity Inova 500spectrometer as described previously (8). The product of theGPDH-catalyzed reduction of GLY by NADH in a reactionmixture containing 10 mM TEA (pD 8.0), 31 mM sodiumphosphite (93% dianion), 5 mM GLY (total), and 7 mMNADH in D2O was identified by 1H NMR spectroscopy. Thereaction was initiated by the addition of 380 L of GPDHin buffered D2O (pD 8.0, 20 mM TEA) to give a total volumeof 1 mL and a final enzyme concentration of 11 M. Asecond 1H NMR spectrum, recorded after reaction for 12 h,showed the disappearance of ca. 80% of the starting GLY

and the appearance of a singlet at 3.53 ppm, which wasshown to be due to the methylene protons of a correspondingamount of ethylene glycol by the addition of an authenticstandard.

The fraction of GLY present in the free carbonyl form inH2O buffered by 10 mM TEA (pH 7.5) at 25 C and an Iof0.12 (NaCl) (1 - fhyd) was determined by 1H NMRspectroscopy using a coaxial inner tube containing D2O andsuppression of the water peak. A value for 1 - fhyd of 0.06( 0.003 was obtained by comparison of the integrated areasof the signals due to the C2 protons of the free carbonyland hydrated forms of GLY, which is the same as a valuefor 1 - fhyd of 0.06 determined previously for the hydration

of GLY in D2O at pD 7.0, 25C, and an I of 0.10 (8).

RESULTS

Initial velocities, Vi, of the reduction of DHAP by NADHcatalyzed by GPDH at pH 7.5 (10 mM TEA), 25 C, and an

Iof 0.12 (NaCl) were determined by monitoring the decreasein absorbance at 340 nm. Figure 1A shows the dependenceof Vi (M s-1) on the total concentration of DHAP in thepresence of 0.10 mM (2) or 0.20 mM (b) NADH and 1.1nM GPDH. The solid line shows the fit of the data to theMichaelisMenten equation with a (Km)app value of 0.24 mMand a Vmax value of 1.42 10-7 M s-1. The latter was used

to calculate a kcat value of 130 s-

1, and a Km value of 0.13mM for the reactive free carbonyl form of DHAP wascalculated using a value for 1 - fhyd of 0.55 for the fraction

of DHAP present in the free carbonyl form (Scheme 3) (17).These data (Table 1) are in agreement with the publishedkinetic parameters for GPDH (18).

There is a ca. 50% decrease in the activity of GPDH (60M) during an 8 h incubation at pH 7.5 (10 mM TEA) inthe presence of 1.2 mM GLY (free carbonyl form, 20 mM

total GLY) and 0.20 mM NADH at 25C and an I of 0.12(NaCl). Figure 1B shows the dependence of the initial

velocity Vi (M s-1) of the reduction of GLY under these

Scheme 3

FIGURE 1: (A) Dependence of the initial velocity, Vi, of the reductionof DHAP by NADH catalyzed by GPDH (1.1 nM) on the totalconcentration of DHAP in the presence of 0.10 mM (2) or 0.20mM (b) NADH at pH 7.5, 25 C, and an I of 0.12 (NaCl). (B)Dependence of the initial velocity, Vi, of the reduction of GLY (20mM total, 1.2 mM free carbonyl form) by NADH (0.20 mM) onthe concentration of GPDH at pH 7.5, 25 C, and an I of 0.12(NaCl).

Table 1: Kinetic Parameters for Reduction of DihydroxyacetonePhosphate and Glycolaldehyde Catalyzed by GPDH and for Activation

by Phosphite Dianiona

substrate activatorKm

b

(mM)kcat

(s-1)kcat/Km

(M-1 s-1)Kd

c

(M)kcat/KiaKb(M-2 s-1)

DHAPd 0.13 130 1.0 106

GLYe (8.7 ( 0.5) 10-3

GLYf HPO32- 8 ( 1 >0.1 4300 ( 700a At pH 7.5 (10 mM TEA buffer), 25 C, and an I of 0.12 (NaCl).

b Values of Km refer to the free carbonyl form of DHAP or GLY.c Dissociation constant for the phosphite dianion activator. dKineticparameters determined from data shown in Figure 1A. e Kinetic parametersdetermined from data shown in Figure 1B. f Kinetic parameters determinedfrom data shown in Figure 2.

Accelerated Publication Biochemistry, Vol. 47, No. 16, 2008 4577

carbanion in water

Documents