new complexes of peroxidases with hydroxamic acids ...hydrazides, and benznmide were made in water,...

THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 248, No. 2, Issue of January 25, pp. 502-511, 1973

Printed in U.S.A.

New Complexes of Peroxidases with Hydroxamic Acids, Hydrazides, and Amides*

(Received for publication, January 6, 1972)

GREGORY R. SCHONBAUM$

From, the Department of Biochemistry, University of Alberta Medical Xchool, Edmonton, Alberta, Canada, and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

SUMMARY

Horseradish peroxidase forms spectroscopically distinct, reversible complexes with hydroxamic acids (R-CO- NHOH), hydrazides (RCO-NHNH2), amides (RCONH2), and oc-hydroxyketones (RCO--CHsOH). Binding of these compounds to the enzyme depends on the polar and steric character of R and the hydrogen bonding capacity of-CO- X-Y (X-Y = NH-OH, NH-NH,, NH-H, CH,OH). Hydroxamate anions and hydrazide cations do not associate with the enzyme. The dissociation constants (IL) for the enzyme-RCOXY complexes span seven orders of magnitude (Kl - 0.3 to 2 x lop7 M), the greatest affinity being shown by compounds with a planar, aromatic R group. This is at- tributed to an interaction of the R moiety at an apoprotein hydrophobic crevice.

Spectrophotometric, electron paramagnetic resonance, and ma.gnetic susceptibility measurements indicate that the association of horseradish peroxidase with hydroxamic acids entails a transition from a mixed spin state of the enzyme to a high spin derivative.

The spectroscopic characteristics of enzyme-RCOXY complexes are similar, suggesting that X-Y substituents do not interact directly with the metal ion of the prosthetic group but perturb its environment. Analogous conclusions were drawn from (a) the parallelism between RCOXY affinities for manganic and ferric peroxidases which does not pertain to ligands (e.g. F-) substituting in the first coordination sphere of the metal ion and (b) the lack of pronounced spectroscopic changes in RCOXY-ferroperoxidase complexes.

The association of peroxidase with hydroxamic acids is competitively inhibited by specific enzyme substrates (hydrogen donors), permitting the evaluation of so far unknown enzyme-(donor) substrate binding parameters (KS). Such a competitive behavior also implies the proximity of RCOXY to the active site.

RCOXY compounds influence heme-linked ionizations and ligand interchange reactions, e.g. they inhibit the formation of alkaline peroxidase and peroxidase-cyanide complex.

With cyanide and RCOXY, ferriperoxidase gives tertiary

* Communicated in part at the 154th Meeting of the -4merican Chemical Societ,y, Chicago (September 1967). This research was supported by grants from the Medical Research Council of Canada (MT-1270) and Life Insurance Medical Research Fund (G-G4-3G).

$ Present address, St. Jude Children’s Research Hospital, Dept. of Biochemistry, Memphis, Tenn. 38101.

complexes. Their formation occurs sequentially:

HCN RCOXY enzyme \ enzyme-cyanide ,

enzyme- cyanide.RCOXY.

The current work originated with the observation that aromatic peracids are exceptionally efficient oxidants of horseradish peroxidase, the 1:l reaction resulting in the formation of compound I and release of the parent carboxylic acid (I). Two interpretations are consonant with these observa- tions: (a) aromatic peracids form a complex with the enzyme which rearranges to compound I concurrently with the scission of the O-O bond, or (b) in its complex with the enzyme, the peracid is rapidly hydrolyzed , giving the physiological oxidant H202 at, or near, the active site. In either case, peracids appear to have a greater affinity for the enzyme than hydrogen peroxide or its monoalkyl derivatives.

Hydroxamic acids (Scheme IA), hydrazides (B), and ol-hydroxyketones (C) are structurally related to and isoelectronic with peracids (D). Like peracids, they are weak acids (2-5) ;

:“\ $ ‘0 si’

. .H\\ NH

R-C, ,’ R-C, ,’

i-i i-i

(A) (a (Cl CD) SCHCMIC 1

they have a planar O---C---X constellation (6-8) ; and they show a propensity for metal binding (9-13) and H-bond formation (14-16). In view of these similarities, it seemed that Compounds ;I t,o C in Schcmr: 1 might serve as excellent probes of the active site, permitting the assessment of alternatives a and b. This proved to be the case. It has now been established that RCOXY compounds (where R is an aliphatic, alicyclic, or aromatic residue, and X-Y = NH-OH, T\‘H-NHZ, CH-OH, etc.) are not hydrolyzed in the presence of H-peroxidasel but form stable, reversible complexes with the enzyme.

1 The abbreviations used are : H-peroxidase, horseradish peroxidase; EPR, electron paramagnetic resonance.

by guest on January 2, 2021http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

503

In this communication we delineate the spectroscopic and magnetic properties of such RCOXY-peroxidase complexes and present a hypothesis on the nature of enzyme-ligand interactions.

In another commm~icntion we shall outline the kinetics of RCOXY oxidation by compounds I and II, showing that hydroxamic acids are extraordinarily efficient reductants of enzyme- peroxide derivatives.

ESPERIMENTA4L PROCEDURE

Ma2erials and dssaMs-Electrophoretically purified H-peroxidase (Lots HPOFF 6113 and 81F) was obtained from Worthing- ton Biochemical Corp. Based on hemin assays (17), its absorptivities in 0.01 M phosphate (pH 6.5) were: (rnM-l cm-l) ~275 32.3, ~403 102.2, 6499 11.2, and ~641 3.21. The amino acid composition of these preparations (1X) corresponds predomi- nantly, in the classification of Shannon et al., to isoenzyme C (19) or, in Paul and Ptigbrand’s classification, to isoenzyme IIIb (20).

Mangnnic H-pero.z~dnsc,-~lallgallic H-peroxidase (21) was prepared by rcconlbinntion of ape-H-peroxidase with manganic prot,oporphgrin in 0.02 11 bornte (pH 5.3) at 2”. The former was derived from the M~orthington enzyme, either by Theorell’s acid-acetone technique (22) or Teale’s acid-butanone technique (23). Manganic protoporphyrin was synthesized by the method of Taylor (24) and recrystallized from dimethyl sulfoxide-tert- butanol (1: 9, v/v).

IIgdroznmic dcids-Most of the compounds used in this study were synthesized in this laboratory, some by courtesy of Dr. J. Wodxk, by standard h!drox~aminolysis of the corresponding carboxylic eaters or acid chlorides (25). C, H, and N am&es of these derivatives, carried out by Galbraith Laboratories, Knox- ville, Term., were within ~0.2% of the theoretical values. Some compounds wcrc checked against commercial preparations available from Hayncs Chrmical Research Corp., Lugoff, S. C. (o-fluorobcnz- and p-h-tlrosSbeilzhydrosamic acids) ; and Raylo Chemicals, Edmonton, .\lbertn, Canada (I-naphtho-, 2-naphtho-, m-iodobenz-, and cyclohexyl hydrosamic acids). 0-Benzopl hydrosylnminc was prepared by Jencks’ method (26). Other highest purity reagents were obtained either from Fisher Sci- entific Co. or from Mdrich Chemica.1 Co. All solutions were made in water distilled twice, once from alkaline permangsnate.

So&ions-Stock polutious of benzhydroxamic acids, benz- hydrazides, and benznmide were made in water, except in the case of wiodo-, I-napht,ho-, 2-naphtho-, and cyclohexyl hydroxamic acids; the latter were dissolved in aqueous alcohol contain- ing 20~~ (v/v) methanol. Ethanol was the solvent for a-hy- droxyacetophenonr. Except for p-hydroxybenzhydroxamic acid, which is autosidizable, weakly acid solutions of hydroxamic acids and hydrazides ma.? be stored at 4” for several days without detectable decomposition (27).

d[easwements of Equilibrium Constants-The formation of enzyme-ligand complexes (HRP .L) was followed spectrophoto- metrically at 410 mu. .W this wave length, the difference in molar nbsorptivities (le,lo) is greatest (Fig. 2), the ligands gener- a,lly do not absorb rind the measurements are most accurate, since 410 am is near t.he absorption maxima of H-peroxidase (403 nm) and its hydroznmic acid complexes (408 nm) (Fig. 1).

The titrations (Figa. 5, ‘T-10) were carried out at 25” by adding 2- to 40-~1 aliquots of the ligand solutions to 2.5 ml of peroxidase (2 to 10 ,uM), by using calibrated microsyringes. The dependence of the equilibrium constant on hydronium ion concentration was examined in 5 111~ acttate, pH 4 to 5; 10 mM 2,2’-dimethyl glutarate, pH 4 to 6; and 20 to 80 IXM phosphate, pH 5.8 to 8.

Borate (30 mllr) was used only in titrations with benzamide, since it forms complexes with hydroxamic acids (28). Over the entire pH range, the equilibrium constants were nearly independent of ionic strength when p - 0.05 or 0.03.

Molar Absorp tivities of H-peroxidase-RCOXY Complexes- Molar absorptivities of H-peroxidase-RCOXY complexes were evaluated in different buffers at 25” with saturating amounts of the titrant. Whenever this method was not applicable, either due to weak ligand-enzyme affinity (benzamide) or to limited titrant solubility (a-hydroxyacetophenone, cyclohexyl hydroxamic acid), the change in molar absorptivities (Ae,) was derived from the linearized form (Equation 1) of the standard equilibrium relationship (Equation 2). This procedure is permissible when the concentration of the enzyme-ligand complex (HRP,L) is relatively small compared to that of the ligand (AT) and when the enzyme-ligand association occurs in 1: 1 ratio.

(1) 1 HRPT AEXZ 1 -c-e---

LT K~(T) A-41 f(lb-)

K 1

= (HRPT - HRP.L) (L, - HRP.0

HRP.L@) (59

For complexes wit’h hydrazides, T = [I + (H+/K,)] (Equation 3); for hydroxamic acids, r = [I + (I(&+)] (Equation 4). In the above equations, li, is the acid dissociation constant of the ligand; HRPT is the total concentration of all ionized forms of H-peroxidase; and Ailk represents t.he observed absorbance change, at. X am, when the solution light path is 1. From the plot of l/AA verBus l/L,, lil and AQ may be evaluated.

Specfrophoton2etric Ueasuremenfs-These measurements were carried out by using a Cary 14 recording spcctrophotometer equipped with a Universal transmission slidewire. Hence, very small absorbance changes could bc accurnt.cly evaluated.

Electron Parczmagnetic Resonance-EPR spectra were taken at 77 and 4.2% w&h n Varian X-band spectrometer (V-4502) equipped with lOO-kHz field modulation.

Magnetic Susceptibility-The experiment’s were performed with an instrument designed by Tasaki et al. (29). Water was used as ca.libration st.andard. The measurements between 0 and -196’ were first made by using H-peroxidase itself (Fig. 4A) and then in t’he presence of equimolar amounts of benzhydroxamic acid (Fig. 4B). The sample volumes were the same in both cases (0.7 ml). 9 nearly complete conversion (-98%) of the enzyme into its benzhydroxamic acid complex was achieved by addit.ion of CBH&ONHOH (0.36 mg).

Potentiometry-The pH values of the reaction media at 22” were measured by using a Radiometer model 25 pH meter equipped with a Radiometer t,ypc GK 2021 C combined calomel glass electrode.

RESULTS

Op/icnl nn.d Jlag?zetic Properties of II-peroxidase-RCONII Y Complexes (where Y = OH, NIIz, If)-The absorbance of horseradish ferriperoxidase between 220 and 1300 nm is drastically alt,ered on ligation t.o RCONHY. Fig. 1 illustrates this effect for benzlq-drosamic acid (R = phenyl, Y = OH), and a similar pattern is seen with hydrazides (Y = NIIZ) and amides (Y = H). In all cases, there is a typical hypcrchromic effect upon the Soret band, coupled to its shift from 403 nm in H-peroxidase to approximately 408 nm in the RCONHY derivatives. In the visi- ble absorpt,ion region, t,he complexes show bands at 503 to 505 nm and 637 to 639 nm, which may be compared to those of the


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

.6

I .-_-II I I I I , b

350 400 4Yo 550 650 7 Wavelength inm)

Wavelength (nm)

FIG. 2. Difference spectrum of H-peroxidase-benzhydroxamic acid complex versus H-peroxidase (0); the concentration of H-peroxidase was 8.35 X lO+ M; the concentration of benzhydroxamic acid was 2.8~ X 1OW M. Inset, absolute spectrum of 2.&, X 10-6 M benzhydroxamic acid (0) compared to the difference spectrum of the complex (0); 0.02 M potassium phosphate buffer (pH 6.3) and 25”.

enzyme at 497 to 499 nm and 641 nm2 (Table VIII). This distinct absorption pattern remains essentially unaltered from - 196 to 40” and between pH 3 and 9.

In the ultraviolet region, the spectroscopic differences become less striking (Fig. 2) ; between 220 and 245 nm, the observed absorbance is nearly equivalent to the sum of absorba.nces con- tributed by the enzyme and its ligand.

The EPR spectra of H-peroxidase and its benzhydroxamic acid complex point to changes in the “heme-linked” interactions. Thus, at 77 and 4.2”K the unliganded enzyme absorbs near g = 6.2 and g = 5.0, respectively (Fig. 3, a and b). On the other hand, at 77°K the absorption derivative of the complex suggests coalescence of transitions giving, near g = 5.95, an in- tense but rather broad band (-220 Oe). The latter is resolved,

2 Spectroscopically analogous complexes are formed with wheat germ peroxidase but not with cytochrome c peroxidase, lactoper- oxidase, chloroperoxidase, sperm whale metmyoglobin, or horse blood catalase.

L

I O(

FIG. 1. Spectra of H-peroxidase (---) FIG. 1. Spectra of H-peroxidase (---) and its benzhydroxamic acid deriva- and its benzhydroxamic acid derivative (-); 0.62 M potassium phos- tive (-); 0.62 M potassium phosphate buffer (pH 6.8) at 25”. phate buffer (pH 6.8) at 25”.

654 3

0 I 2 3 4 0 1 2 3 A

Magnetic Field ikoe:

FIG. 3. EPR spectra of H-peroxidase (a and 5) and its benzhydroxamic acid complex (c and d) at 77 and 4.2’K; 0.6 mM H-peroxidase in 0.1 M potassium phosphate buffer (pH 6.8). EPR spectra were measured, under the same conditions, before and after addition of an equimolar amount of benzhydroxamic acid. Microwave frequency, 9.02 GHz; modulation amplitude, ~8 Oe.

at 4.2”K, into a doublet with g - 6.1 and g - 5.4. The difference between the splitting factors is substantially smaller in the complex (Ag = 0.7) than in the free enzyme (Ag = 1.2) and implies a transition to a higher axial symmetry of the coenzyme (30) or reflects a change in the spin relaxations.

Magnetic susceptibility measurements complement the above data (Fig. 4). For H-peroxidase, the effective magnetic moment (nerf) is 5.23 Bohr magnetons, which is in excellent agreement with the results of Theorell and Ehrenberg (31). In the presence of benzhydroxamic acid, the paramagnetism increases, giving n,fr of 5.97 Bohr magnetons. This is effectively the same as the theoretically computed value for an ion with five unpaired electrons (n,tf = 5.92 Bohr magnetons). The magnetic suscepti- bilities of H-peroxidase and its benzhydroxamic acid complex follow Curie’s Law, between - 196 and 0’. Apparently, in contrast to several other hemoproteins (29, 32-34), both free and liganded forms of the H-peroxidase retain their high spin character over a wide temperature range.

Dissociation Constants of H-peroxidase-RCOX Y Derivatives-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

-50 ("Cl 0 j -100 -150 -180 -200

103/T (“K-l)

FIG. 4. Temperature dependence of molar paramagnetic susceptibility of 3.73 mu H-peroxidase (A) and its benzhydroxamic acid complex (B). Benzhydroxamic acid, 3.7 mM; 0.01 M potassium phosphate buffer (pH 0.5).

0.6 106K,, M’ -

T E

2

E c 0.5

:

i5 / ? 225

0 IO 20 IO 20

10’ Benzhydroxomic acid (M) 7+ log (BHA,-HRP.BHA)

FIG. 5. Titration of 4.1 X 1OW M H-peroxidase with benzhydroxamic acid; 0.02 %I pobassium phosphate buffer (pH 6.0) at 25”. The apparent dissociation constants (Kl) were computed from the data at indicated points. Znsel, log-log plot, based on Equation 2; the slope of the line is 1.

The assay of the enzyme-RCOXY complexes was based on the spectroscopic measurements described in detail under “Experi- mental Procedure.” In all cases, the equilibrium was established within the mixing time of the reagents. Between pH 4 and 9 at 25”, the complexes are stable and no secondary derivatives were detectable. At all levels of enzyme saturation, the spectra pass through a single set of isosbestic points.

A typical experiment. for the measurement of benzhydroxamic acid binding is illustrated in Fig. 5. The data and the slope of 1, derived from the logarithmic plot of Equation 2, establish that the reactants combine in 1: 1 ratio. The same holds for Ohe other complexes listed in Table I.

In evaluating Zil, it is essential to take into account the acid- base properties of t’he ligands (2-4) (Equations 3 and 4).

RCONHNHa+ 4 RCONHNH, + H+ (PK, = 3.3) (3)

RCONHOH + RCONHO- + H+ (pK, = 8.8) (4)

505

TABLE I Apparent dissociation constants (K,) of peroxidase-hydroxamic acid

complexes in 0.02 M potassium phosphate bu$er (pH 6.0) at 25”

Hydroxamic acid 106 K,

M

2-Naphtho- 0.2

Benz- 2.4

Isonicotino- 100

l-Naphtho- 22.0

p-Methylbenz- 2.6

o-liluorobenz- 10.5

p-Fluorobenz- 13

m-Fluorobenz- 22

Cyclohexyl- 2400

-

p-Methoxybenz. 7.5

p-chlorobenz- 6

m-Chlorobenz- 4

Aceto- 62,000

-2

c

>-Hydroxybenz- 9.0

p-Hydroxybenz- 7.8

m-Iodobenz- 2.2

Form- 3 x 106

TABLE II Efect of pH on the apparent dissociation constants of peroxidase-

benzhydroxamic acid, -ben.zhydrazide, and -benzamide complexes at 26’

Complex

Benzhydroxamic acid (pK, = 8.8)

Benzhydrazide (pK,b = 3,3)

Benzamide

PH

4.11 5.05 5.98 6.82 8.75

3.55 4.12 5.05 6.10 6.74 7.3

3.59 4.26 5.98 6.82 8.87 9.56

1C ‘$ Kobr

Apparent dissociation constant

2.45 3.77

1.35 2.51 2.35 3.42 !.lO

M

2.0 1.38

1.3 1.2 0.87 0.98 0.92 1.0

3.5 3.3 3.6 3.4 3.3 3.1

a K,,bobs = K, X r (Equation 2). b For the conjugate acid form CgH.&ONH-NH3+ (Equation 3).

For any given compound then, K1 calculated on the basis of the neutral form is virtually invariant from pH 3.5 to 9.0 (Table II). Amides, which do not ionize or protonate between pH 1 and 14, associate with the enzyme equally well in acid and alkaline solutions (Table II), providing that the conversion of neutral H- peroxidase into its alkaline derivative (characterized by pK, - 10.9 (35)) is taken into account. This is expressed in Equation 5.

log AA K&I

AAT - AA = pH + log ___

LT + KI (5)

where LT = (benzamide) >> (enzyme). Ad represents the observed change in absorbance at 395 nm, a wave length isosbestic


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

506

12.0

11.5

11.0

PH

0 0.5 1.0

log (1 t Benzamide

K, 7

FIG. 6. a, inhibition of alkaline peroxidase formation (peroxidase-hydroxide complex) by benzamide. b, analysis of the results shown in Fig. Ga, according to Equation 6. H-peroxidaser, 7.25 PM; benzamider, 5.4 mrvr (o), 16.1 mM (A), 34.2 mM (0) and 68.2 mM (0) at 25”.

for the neutral form of H-peroxidase and its benzamide complex; AAT is the maximum change in absorbance attending conversion of the enzyme into its alkaline derivative.

Analyses of H-peroxidase-benzamide interaction, shown in Fig. 6a, are consonant with the predictions based on Equation 5. Thus, at different benzamide concentrations, the plots of log (AA/AAT - AA) versus pH give lines of slope 1; and the pH at which log (AA/AAT - AA) = 0, i.e. pKacapp), increases with higher concentrations of the ligand. This is illustrated by the secondary plot shown in Fig. 6b, where

~K.wp) = PIG + log (1 + -WC) u3

The intercept on the ordinate gives pK, - 10.95, in excellent agreement with the value obtained through direct titration of the enzyme (35).

Three conclusions are allowed by the above data. (a) The heme-linked effect leading to the formation of alkaline peroxidase is governed by a single ionization; (b) below pH 12, H-peroxidase- benzamide complex is not subject to such an ionization; and (c) if benzamide binds to the alkaline peroxidase, then its affinity is much weaker than that for the neutral enzyme.

Interaction of H-peroxidase with Cyanide in the Presence of Hydroxamic Acids and Hydrazides-If the enzyme-RCOXY interaction involves a direct coordination of -COXY to the ferric ion, such a binding should be competitively inhibited by typical hemoprotein ligands such as cyanide, fluoride, or aside. Of these, cyanide is the reagent of choice. It reacts readily with H-peroxidase (36, 37), giving a complex whose dissociation constant is nearly pH invariant from pH 4.2 to 7.5 (37) and whose spectrum (38, 39) differs radically from those of H-peroxidase- RCOXY derivatives (Fig. 1).

For a fully competitive ligand interchange in a peroxidase- benzhydroxamic acid-cyanide system, the ratio (HRP . HCN) : (HRPeBHA) should be

HRP.HCN K1 HRP.BHA = K, ’

HCNT BHAT (7)

providing that the concentrations of ligands BHAz and HCh-z are at least IO-fold greater than the total concentration of the enzyme, HRPT. K1 and KS are the dissociation constants of peroxidase-RCOXY and peroxidase-cyanide complexes. The above relationship expressed in linear form

1 1 Kl HRP . BHA

=-------x-x HRPT KS

B+ X HCNT -I- T H& C8)

lo- b.

0 250

106x HCN (M)

1 FIG. 7. a, assay of peroxidase-benzhydroxamic acid complex

(HRP.BHA) in the presence of cyanide (HCN). Peroxidase- cyanide complexes were determined at 428.5 nm (X isosbestic for peroxidase and HRP+BHA; Ae = 67 X 10Z Ma1 cm-r). HRP.BHA was evaluated at 412.5 nm (X isosbestic for peroxidase and peroxidase-cyanide; -AE = 53 X 108 M-I cm-l). PeroxidaseT, 8.3 PM; benzhydroxamic acidr: 0.097 mivr (O), 0.194 mM (Cl), 0.385 mM (0), and 0.580 mM (A); 0.05 M phosphate (pH 6) at 25”. The calculated slopes for a fully competitive ligand interchange are illustrated by dashed lines, when BHAT = 0.095 mM (upper line) and BHAr = 0.38 mM (Lower line). b, analysis of the data in Fig. 7a according to Equation 12.

TABLE III

Titration of H-peroxidase-benzhydroxamic acid complex with cyanide

Conditions: (II-peroxidasez) 8.3 PM; 0.05 M potassium phosphate (pH 6.05) at 25O.

10s BHA I

10-o m I

I KdKa(sppP

M

97 194 385 580

a KJKa ccalo) = 1.24.

bf-2

2.18 1.75 1.60 2.58 1.20 3.84 1.10 5.29

implies that m x HRPz x BHAT = Kl/KB = 1.25, where m = slope of l/HRP.BHA versus HCNr plot; K1 = 2.4 x 1W6 M (Table I), and KS = 1.9 f 0.2 X 10Wm (37, 40).

The results shown in Fig. 7a do not accord with this analysis. Instead, we note a progressive increase in t.he partition constant, KI/K3, with increasing concentration of benzhydroxamic acid (Table III).

I f this trend reflects the formation of a ternary H-peroxidase- cyanide-benzhydroxamic acid complex (Equation 9),

K 4

= (HRP.HCN)(BHAd HRP.HCN.BHA (9)

the relationship between l/HRP.BHA and HCNT should be

1 1 KI = -X-X(&++) HCh+& (lo)

HRP.BHA HRPT KS

The slope, m, of l/HRP .BHA versus HCST plot now becomes

m=&X~X(&+$) T

(11)


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

507

Hence, these requirements is

(12)

Hy using Equation 12 and the data shown in Fig. 7b, Kq was found to be 1.5 =t 0.1 X 10W4 M and K1:K3 = 1.11 f 0.05, in agreement with the espccted K1 : Ks value.

The results of similar analyses, using l- and 2-naphthohydroxamic acid and benzhydrazide are summarized in Table IV. In all cases, the observed partition constant, K1:K3, closely agrees with Kl:Ksccalc) by using RI and Ks constants obtained in independent studies. Furthermore, according to Equation 11, m should converge to a limiting value with increasing ligand (I&) concentration and become equivalent to (~/HRPT) X (K1/K3) x (l/K,), when l/L, < l/Kd. Under such conditions, the extent but not the rate of formation of H-peroxidase-cyanide complexes (5 E. Cx + E. CN L) should be nearly independent of LT. And such is t,he case (Table V).

The data presented in Table V indicates that the initial velocity (v;) of format,ion of the enzyme-cyanide complexes (E . CN -+- E. CN . BZH) is proportional to the concentrations of cyanide (HCNT) a.nd free enzyme (Ef). The simplest scheme meeting

TABLE IV Apparent dissociation consta.nts of ternary peroxidase-cyanide-

ligand complexes in 0.06 M phosphate (pH 6) at .W’

Ligand 10’ X Kd

M

2-Naphthohydroxamic acid. 0.11 1-Naphthohydroxamic acid. 1.1 Benzhydroxamic acid.. . 1.5 Benzhydrazide 4.2 Fluoride@. . .

0.11 11 1.1

45 2.2 x 103

0.10 10

1.2 50

2.2 x 103

a In separate experiments, it was shown that the dissociation constant (KI) for the peroxidase-fluoride complex (expressed in terms of t.he tot,al concentration of reagents) is: K1 = 4 X 10e3 M,

at pH 6 a.nd 25”.

TABLE V

Rates and extent of formation of peroxidase-cyanide complexes in the presence of benzhydrazide (BZH)

Conditions: (H-peroxidaser) 4.4 X 1Om6 M; 0.05 M potassium phosphate (pH G) at 25’.

BZH HCNT ECN T’- XL103 x 106 , X106

M M-1

7 1 1.3 0.45 1.25 f 0.05 14 1.3 0.45 1.25 f 0.05 21 1.3 0.44 1.25 4~ 0.05 t:

( 2.5 5.0 0.81 1.41 1.25 1.25 f f 0.05 0.05

_~

M M s-1

6.2 1.0 3.1 0.55 2.1 0.33 2.1 0.64 2.1 1.39

M-1 s-1

1.2 1.4 1.2 1.2 1.3

- a ECNT, equilibrium concentration of all forms of enzyme-

cyanide complexes, determined from the absorbance changes at 429 nm.

* Kl/Kar = m HRPT (Equation 11). 6 Ef = EFK,/(LT + KI). d Initial velocity of ECNT formation, vi = AA/Ae X l/At, where

AC = 66 X 1Oa M-l cm-l and AA is the change in absorbance at 429 nm within 10 s of cyanide addition.

e ks, the apparent rate constant derived from Equation 13.

WJ E .---- E.L’

1 , MHCN

and

d(E.CN) Vi=T+

d(E.CN-L) dt

= K% X HCNT (13) 1 T

k3 evaluated in this manner (Table V) was found to be: 1.3 + 0.1 X lo5 M-’ s-l, compared to & = 1.0 f 0.1 X lo5 M-~ s?

obtained by Chance (36) and Ellis et al. (37). Direct Determination of K4--Direct evaluation of KJ became

possible with the observation that the spectrum of the enzyme- cyanide complex is slightly different in the presence and in the absence of RCOXY ligands.

Such changes are independent of cyanide concentration when K1 HCNT >> KBLT. The maximum differences in molar absorptivities (eHRP.HCN.RCOXY - cHRPsHCN) range from approximately AEJ, = 3000 M-* cm-l when RCOXY = 2-naphthohydroxamic acid to approximately Ae~so = 5000 M-’ cm-l with benzhydrazide (Fig. 8a). S’ mce at 430 nm the absolute molar absorptivity of H-peroxidase-cyanide complex is 82 f 1 x 1Oa ~-1 cm-l, it is evident that the increase induced by RCOXY compounds is relatively small. Nonetheless, this is adequate for an accurate measurement of RCOXY binding to the enzyme- cyanide complex.

Accordingly, from the data presented in Fig. 8a, we obtain K( = 4.4 x 10W4 M in good agreement with the value derived indirectly (cf. Table IV).

Interaction of H-peroxidase with Hydrogen Donors-Although phenols and aromatic amines have long been recognized as the specific peroxidase substrates (41), the association constants governing their interaction with the enzyme are unknown. Such parameters can be now readily calculated, exploiting the inhibition of RCOXY binding in the presence of hydrogen donor substrates. This is illustrated in Fig. 9A. From these results, we can derive the dissociation constants (KS) for H-peroxidase- substrate (HRP.8) complexes, assuming that binding of the specific substrates (8) and titrants (L) occurs at the same enzyme site. Wit.h substrate in excess (ST >> HRPT) at a pH where S and L are largely in the unionized forms, the relevant relationship is:

HRP.L HRPT K LT - HRP,L

=-xx- KS Kl ST + Ks KI@T + 9,)

(14)

X HRP.L

from which K, may be obtained. Plots based on Equation 14 are shown in Fig. 9B for benzhydrazide, both in the absence of substrates and in the presence of 2.3 mM hydroquinone, 5.2 mM aniline, and 9.1 mM phenol. In all cases, the lines intersect the abscissa at a point corresponding to the total enzyme concentration. Also, as would be expected for competitive binding, the calculated dissociation constants are independent of the initial hydrogen donor concentration or the nature of the titrant (Ta- ble VI).

Apparently, in contrast to the association between RCOXY and H-peroxidase-cyanide complex, the binding of hydrogen donors and RCOXY is fully competitive. An independent


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

508

r T

, b. a

’ ///

P

d

.

/*

lOax Benrhydrozide,

v+l

FIG. 8. a, difference spectra obtained on Ctrating 4.2 PM peroxidsse- cyanide complex with benxhydrazide. HCN=, 3 mM; 0.08 M phosphate (pH 6) at 25O. b, analysis of the data in Fig. 8a according to Equation 1. HCNT, 3 mM (0); 14 rn>¶ (0).

\ 7 16.3

-1 0 I

A50 440 430 420 410

Wavelength (nm)

1 !

0 100 200 300

l/‘AA 130

Interaction of RCOX Y with Manganic Peroxidases and Ferro-

peroxidases-The absorbance pattern of Mn(II1) protoporphyrin apoperoxidase is altered on formation of the enzyme-hydroxamic acid derivatives; the greatest differences being associated with r-z* transitions of the porphyrin macrocycle (Table VII). The dissociation constants, K1, derived in this manner are 2.1 f 0.2 X 10e6 M for the benzhydroxamic acid complex and 1.6 -or 0.2 X 1O-7 M for the 2-naphthohydrosamic acid derivative. For the corresponding complexes with the ferric enzyme, the dissociation constants are 2.4 X lop6 M and 2 X 10e7 M. Clearly, the affinities of peroxidases for the aromatic hydroxamic acids are not governed by the identity of the central metal ion. This is by no means typical of Mn(III)- and Fe(III)-enzyme reactivi- ties (21, 42).3

On the other hand, RCOXY compounds do not bind equally well to the reduced enzyme and, as shown in Table VII, do not elicit large spectroscopic changes. Such weak heme-linked effects are analogous to those observed with peroxidase-phenol or peroxidase-cyanide-RCOXY complexes (Figs. 8 and 10) and indicate that the -COXY residue does not coordinate to the metal ion. The dissociation constant of ferroperoxidase-bens- hydroxamic acid complex is approximately K1 = 3 f 2 x 1OW M at pH 7.1 (in 5 mM phosphate) and 25”; i.e. it is of the same order of magnitude as Kd for the ferriperoxidase-cyanide- benzhydroxamic acid complex (Table IV). The uncertainty in K1 is largely due to the autoxidizability of the ferrous enzyme. Thus, the spectrophotometric measurements used in these assays are subject to errors which, so far, we have not been able to eliminate.

DISCUSSION

The RCOXY ligands may be divided into two categories. In one subclass we have hydroxamic acids, hydraaides, amides, and cu-hydroxyacetophenone, all of which associate with H-peroxidase to give spectroscopically distinct derivatives. The second group includes IV- and O-substituted hydrosamic acids, 0-benzoyl hydroxylamine, 4 N - hydroxybenzene sulfonamide, phenacyl halides, or benzaldehydc which, like phenols and aromatic amines, appear to bind to the enzyme but without markedly changing its characteristic spectrum. In some cases, although not with

a Unlike those for RCOXY derivatives, the dissociation constants for manganic and ferric fluoride complexes differ approximately 40-fold, the manganie enzyme showing a weaker affinity.

4 0-Benzoyl hydroxylamine reacts slowly with H-peroxidase forming, in the initial stages of the reaction, compounds which are spectroscopically similar to the enzyme-peroxide derivatives.

0 20 40 60 80 0 1 2

lo* Benzhydrazide (MI lo6 HRP-BZH (Ml

FIG. 9. A, tit.ration of 4.1 X lo+ M H-peroxidase wit.h benzhy- &aside, in the absence (0) and presence of hydrogen donors: 5.2 mM aniline (0); 2.3 mM hydroquinone (0); and 9.1 rnM phenol (m). B, results shown in A, replotted according to Equation 14. BZH, benzhydrazide.

TABLE VI

Apparent dissociation constants of peroxidase-hydrogen dolwr complexes, evaluated by using Equation 14, in 0.08 N potassium

phosphate bu$er at 25” - -

-

-

_ _

-

Substrate

COnCEXl- tration range x 10’

M

2-10

2-10 4-12

2.5-7.5 2-6

Titrant X 101 K,

.a*

4.3 f 0.2

4.2 zk 0.2 17.7 * 1.3 16.4 f 1.0 2.5 f 0.3

Benzhydroxamic acid

Benzhydrazide Benzhydrazide Benzhydrazide Benzhydrazide

6.8

6.8 6.7 6.7 6.7

Phenol

Phenol Aniline Mesidine Hydroqui-

none -

evaluation of Kphenor by analysis of the difference spectrum between H-peroxidase and its phenol complex is shown in Fig. 1Oa. From the observed hyperbolic increase in absorbance with increasing concentration of phenol, Knhenol was computed to be 5.0 =I= 0.5 X lop3 M (Fig. 10~). Furthermore, the results in Fig. lob demonstrate that phenol associates nearly as well with ferriperoxidase-cyanide complex. Thus, gauged by phenol binding the conversion of high spin ferric enzyme into a low spin cyanide derivative does not entail a pronounced change in the structure of the apoenzyme.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

509

400

AdO 430 420 410 400 0 200 400 600

Wavelength (nm) 1 ‘AA

TABLE VII

Effect of benzhydroxamic acid and benzhydrazide on the absorption spectra of manganic peroxidase and ferroperoxidasea*C and

ferroperoxidase-carbon monoxide complexesb~c

CHOH) appears unlikely, because (i) cY-hydroxyacetophenone enolizes slowly (ti/~ - 170 hours) (44)) whereas its ligation to the enzyme is fast (ti/* < 10 s, when LT = 3 mM); (ii) imidic acids, enol forms of amides, are unknown (45) ; and (iii) in the 220 to 250 nm range the difference spectrum of the enzyme-“benzhydroxamic” acid complex and H-peroxidase (Fig. 2) is superim- posable on the absolute spectrum of the ligand. Contributions from the enol tautomer, benzhydroximic acid, would be expected to show an altered absorption pattern (cf. the differences in the spectra of 0-alkyl benzhydroxamic and benzhydroximic acids (46)).

Peroxidase Ti trant Absorption bands

Manganic

Manganic

Ferro-

Ferro-

Ferro-

Ferro. CO

Ferros CO

FerroeCO

Benzhydroxamic acid

Benzhydroxamic acid

Benzhydrnzide

Benzhydrazide

Benzhydroxamic acid

x (nm)/t @m-l cm-9

482 376 42.8 80.7

476 375 53.8 73.8

557.2 437.1 13.1 89

556 435 81

557 428 12.9 94.4

572.5 541.6 422.9 13.4 13.9 161

571 542.6 424 12.7 14.9 155

423 161

a The enzyme was reduced under argon with N&&O,. 5 In the presence of approximately 1 mM CO. c In 5 rnM phosphate, at pH 7.1, and 25”.

benzaldehyde and O-benzoyl hydroxylamine, this behavior may be due to inherent steric demands of a given substitutent and may also reflect conformational isomerism, dictated by the re- pulsion of nonbonding electrons on X-Y and the a-electrons of the carbonyl group (7, 43).

In this context, the following points deserve emphasis: (a) Ionized ligands (benzhydrosamate anion, benzhydrazide cation) do not associate with H-peroxidase (Table II). This is consistent with, but does not establish, the nonpolar nature of the -COXY binding site. (b) The carbonyl group is not the sole determinant in the formation of spectroscopically active enzyme complexes. For example, benzaldehyde is an inert titrant. (c) When X = NH, Y can be -OH, -NH2, or -H; and when Y = OH, X can be -NH or -CH2. Hence these groups per se cannot play a dominant role in coordination. (G?) Association of the enzyme with the enol tautomers (e.g. R-C(OH)=NOH, R-C(OH)=

FIG. 10. a, difference spectra obtained on titrating peroxidase with phenol. H-peroxidaser, 4.2 1~; 0.08 M phosphate (pH 6) at 25”. 6, difference spectra resulting on titrating peroxidase-cyanide with phenol. H-peroxi- daseT, 4.2 pt~; 0.08 M phosphate (pH 6) at 25”; HCNr, 3 mM. c, results shown in a and b, replotted according to Equation 1.

We infer that spectroscopically distinct complexes are only formed when the ligands contain an acidic residue (X), in a for- mally uncharged -C-X-Y constellation. This underscores

II 0

the import,ance of the functional group as a whole rather than the individual components. Mechanistically, this may imply either chelation of the hemin-iron or polyfunctional H-bonding.

The chelation hypothesis is less likely on at least two counts. First, it is hardly plausible for amides; second, it is difficult to reconcile with the kinetics of RCOXY binding. For instance, with benzhydroxamic acid, the apparent second order rate constant (k,,, - 0.4 f 0.1 X lo* M-~ s-l at 25’)s is greater than for any other H-peroxidase-ligand reaction.

Polyfunctional hydrogen bonding, resembling the hydrogen- bonded interactions of ureido groups with proteins (47, 48), is not open to these objections. It is supported by a correlation between peroxidase-ligand affinity (expressed by K i) and acid dissociation constant of the ligands, K, (Fig. II). Such rela- tionships (49-52) rest on an intrinsic property of the hydrogen bond, relating its strength to the tendency of proton transfer (expressed by K,) from the donor-acid to the acceptor-base.

Accordingly, in a homologous series of compounds (e.g. R = C6Hs), we expect the greatest affinity between peroxidase and benzhydroxamic acid (pK, 8.8) (2, 3), decreasing with benzhydrazide (pK, - 11.5 to 12.5) (4), and even more so with benzamide (pK, - 15) (10). Such a trend is discernible in Fig. 11. For c+hydroxyacetophenone, K, is unknown but, like that of a-haloacetophenones, it should be lo-i6 to 10-i’ M (53). Hence, the ketol would be a weak ligand and such is the case.

Hydrogen bonding, particularly of the monofunctional type,

5 B. Chance and G. R. Schonbaum, manuscript in preparation.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

510

1, 2- cY.Hydraxyocetophenone m /

tkzamide /

u 3-

2 I 4-

/ w Benrhydrozide

5- / 6- /

. Benzhydroxomic acid

5 10 15 20

PL

FIG. 11. Log-log plot of H-peroxidase-RCOXY dissociation constants (K1) versus the corresponding ligand acidities (K,).

TABLE VIII Spectroscopic characteristics of H-peroxidase complexes in 10 rnM

potassium phosphate bu$er (pH 6.5) at .25O

Ligand

None

2-Naphthohydrox- amic acid

Benzhydroxamic acid

Aoetohydroxamic acid

Benzhydrazide

Isonicotinic acid hydrazide

Benzamide

Absorption bands X(nm)/lO-a*

-

641 3.21

638 3.71

638 3.77

638 3.57

639 3.47

639

M-1 cm-’

499

11.3 504

10.4 504

10.4 503

10.5 503

10.9 503

639 502 3.42 10.9

403 102.2 408 154 408 152 407 151 407 141 406 130 407 126

r

I .-

)ifference in molar absorptivities

:H&Y)~p)

Al-1 m-l

60

59.5

55

46

35

28

does not provide large binding energy (54) but becomes more significant when polyfunctional interactions can occur (55).

Consider now the following equilibria

HRP.L’

“I/ K’y.

K’ = (HRP)(L)/HRP-L K” = HRP.L/HRP.L’

Peroxidase + L - HRP*L

I f HRP.L’ is the only enzyme-ligand derivative, then ligation of benzamide, benehydrazide, and benzhydroxamic acid are defined by A6410 values ranging from 28 x lo3 to 60 X lo3 M-I cm-1 (Table VIII), a gradation of perturbation effects dependent on the -COXY structure.

Alternatively, if a spectroscopically inert HRP .L complex precedes the formation of HRP.L’, then Ae will be contingent on K”. When K" is Iargc, only small spectroscopic changes may be expected. K' is then analogous to Krq, as defined for ternary enzyme-ligand complexes. Such comparison strengthens the proposal that binding of the R group of RCOXY is the main driving force in the ligand-enzyme association. It is this type of interaction, the hydrophobic binding, which must be responsi- ble for the higher affinity of 2-naphthohydroxamic acid (K1 - 2 x low7 M), compared to that of acetohydroxamic acid (K1 -

620,000 x lo-’ M) and formhydroxamic acid (K1 - 0.3 M). The change in free energy of binding from 2-naphtho- t.o formhydroxamic acid amounts to -8 kcal per molt, i.e. within the range of binding energies (-5 to - 10 kcal per mole) observed on association of nonpolar compounds with proteins (56, 57).

A plausible hypothesis is that both R and -CO-X-Y residues

R-CNHY L - - -Fe-L’ e L--Fe--L’..MN

associate at a heme-linked, extended, largely apolar region of H-peroxidase, in which the R-crevice is also the binding site for the hydrogen donor substrates. The association of the -COXY moiety near the prosthetic group with a concomitant change in the relative position of trans.ligands, the central metal ion, and porphyrin would give a configuration more akin to that of metmyoglobin, where the porphyrin ring is not. planar but slightly concave towards the sixth coordination position (58). The lower rhombicity of the coenzyme (Fig. 3) and the higher paramagnetism of the H-peroxidase-benzhydroxamic acid complex (Fig. 4) and its metmyoglobill-like optical spectrum (Fig. 1) support this argument.

The inhibition of alkaline peroxidase format,ion by benzamide (Fig. 6) also accords with the proposed model, since both the heme-linked ionization of the Fe- -L’ . . 9 HX system and ligand interchange reactions are impaired in the ferriperosidase-benzamide complex.

Furthermore, the similar affinities of mangarlic and ferric peroxidases for hydroxamic acids cont,rast with those for fluoride and suggest again that the formation of RCOKHOH-enzyme complexes does not entail replacement of a ligand in the first coordination sphere of the metal ion. Rather, as outlined previously, CO-X-Y exerts its effect indirectly, through hydrogen bonding, most likely to water coordinated at the distal site of the prosthetic group (L’H=H*O). Such hydrogen bonding should be of lesser importance when cyanide occupies the distal site since the nitrile nitrogen is only a very weak proton acceptor (59). Indeed, the differences in affinities of ferri- peroxida.se-cyanide for benzhydroxamic acid and benzhydrazide (Table IV) are much smaller than those for the corresponding complexes with ferriperoxidase itself (Table I).

Similarly, if in ferroperoxidasc the sist,h coordination site of the prosthetic group is not occupied by water, as in ferromyo- globin (60), the dissociation constants for ferroperoxidase- RCOXY complexes should parallel lid rather than K1 values. The preliminary data on ferroperosidnse-benzhydrosamic acid complex, where K = 3 + 2 X 10-4~ supports this proposal.

These results do not preclude some int,eraction between the metal ion and the COXY moiety. For thr observed shift of the ferroperoxidase Soret band from 437 to s-428 nm, which is caused by an amino reagent, benzhydrazide, but not by benzhydroxamic acid (Table VII), may indicate a, weak contribution from a hemochromogen type of complex. However, its formation cannot be extensive since the changes in the Soret region are not reflected in the pattern of cy- and P-bands.

The strongest evidence suggesting the proximity of the RCOXY site to the prosthetic group is the fully competitive binding between RCOXY ligands and t,he peroxidase H-donors, i.e. phenols and aromatic amines (Table VI, Figs. 9 and 10).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

This, and the exceptional reactivity of hydroxamic acids5 zf. towards Compounds I and II are difficult to reconcile with alter- . native schemes whereby RCOXY causes a conformational 2%. change transmitted to the hemin environment from a remote binding site. 29.

Ac~rwwledgmentsI am indebted to Dr. A. Tasaki and Dr. T. 30.

Asakura for their collaboration in EPR and magnetic susceptibility measurements, and to Mrs. L. Gomez-Rao for her excellent 31. assistance. Thanks are due to Dr. P. Nicholls for helpful dis- 32. cussions. 33.

1.

2.

3.

4.

5.

6.

;:

9.

10.

11. 12. 13.

14.

15. 16.

17.

18.

19.

20.

REFERENCES 34. SCHONHAUM, G. R., AND Lo, S. (1972) J. Biol. Chem. 247,3353-

3360 GREEN, A. L., SAINSBURY, G. L., SAVILLE. B., AND STANS-

FIELD, M. (1958) J. Chem. Sot. 15X3-1592 WISIC, W. M., AND BRANDT, W. W. (1955) J. Amer. Chem. Sot.

77, 1058-1059

35. 36. 37.

TAMURA, Z., AND NAGANO, K. (1963) Chem. Pharm. Bull. (Tokyo) 11, 793

38. 39. 40.

HOMER, R. B., AND JOHNSON, C. D. (1970) in The Chemistry of Amides (ZABICICI, J., ed) p. 187, Interscience Publishers, N. Y.

JENSEN, L. H. (1954) J. Amer. Chem. Sot. 76, 4663; (1956) J. Amer. Chem. Sot. 78, 3993

EXNER, 0. (1965) Collect. Czech. Chem. Comm. 30, 652 SMITH, P. A. S. (1965) Open-Chain Nitrogen Compounds, Vol.

1, p. 139, W. A. Benjamin Inc., N. Y. SCHWARZENDACH, G., AND SCHWARZENBACH, K. (1963) Helv.

Chim. Acta 46, 1390

41.

42.

43.

44.

45. SMITH, P. A. S. (1965) Open-Chain Nitrogen Comwounds. Vol. ANDEREGG, G., L’EPLATTENIER, F., AND SCHWARZENBACH, G.

(1963) Helv. Chim. Acta 46, 1400 DUTTA, R. L. (1959) J. Indian Chem. Sot. 36, 285 NEILBNDS, J. B. (196G) Struct. Bonding 1, 59 NAGANO, K., KINOSHITA, H., AND TAMURA, Z. (1963) Chem.

Phamn. Bull. (Tokyo) 11, 999

46. 47. 48. 49. 50.

HADZI, D., AND PREVOSEIC, 0. (1957) Spectrochim. Acta 10, 38 51.

JENSEN, J. B. (1956) Acta Chem. &and. 10, 667 BROWN. H. C., AND PILIPOVICN, D. (1960) J. Amer. Chem. Sot.

82, 47004703 52. 53.

P.~~JL, K. G., THICORE:LL, H., lick APXSON, A. (1953) Acta Chem. &and. 7, 1284

WELINDER, K. G., SMILLIE, L. B., AND SCHONBAUM, G. R. (1972) Can. J. Biochem. 60, 44

SHANNON, L. M., KAY, E., AND LEW, J. Y. (1966) J. Biol. Chem. 241, 2166-2172

54.

55.

PAUL, K. G., AND STIGBRAND, T. (1970) Acta Chem. &and. 24, 5G. 3607 57.

21. GJESSING, E. C., AND SUMNER, J. B. (1942) Arch. Biochem. 1, 58. 1

22. THEORELL, H. (1941) Ark. Kemi Mineral. Geol. 14B, 20 23. TEAL>:, F. W. J. (1959) Biochim. Biophys. Acta 35, 543 59. 24. TAYLOR, J. J. (1940) J. Biol. Chem. 136, 569 25. HAUSER, C. R., AND RXNFRXW, W. B. (1943) in Organic Syn-

theses (BLATT, A. H., ed) Collect. Vol. II, p. 67, John Wiley 60. & Sons, N. Y.

511

JENCKS, W. P. (1958) J. Amer. Chem. Sot. 80, 45814584 SMITH, P. A. S. (1966) Open-Chain Nitrogen Compounds, Vol.

2, p. 79, W. A. Benjamin Inc., N. Y. F&L&, A. R., DAYE; B. M., A& CHRISTIAN, R., JR. (1966)

Talanta 13, 929 Tasa~r, A., OTSUICA, J., AND KOTANI, M. (1967) Biochim. Bio-

phys. Acta 140, 284-290 PEISACA, J., BLUMBERG, W. E., OGA~~, S., RACHMILEWITZ,

E. A., AND OLTZII~ R. (1971) J. Biol. Chem. 246, 3342-3355 THEORELL, H., AND EHRENBERG, A. (1952) Arch. Biochem.

Biophys. 41, 442 IIZUKA, T., AND YONETANI, T. (1970) Advan. Biophys. 1, 157 Ixzu~a, T., AND KOTANI, M. (1969) Biochim. Biophys. Acta

194, 351-363 IIZUKA. T., AND KOT~~NI, M. (1969) Biochim. Biophys. Acta

181, 275-286 THEORELL, H. (1942) Enzymologia 10, 250 CHANCE, B. (1943) J. Cell. Comp. Physiol. 22, 33 ELLIS, N. B., -END DUNFORD, H. B. (1968) Biochemistry 7,

2054 CHANCE, B. (1949) Arch. Biochem. 21, 416 KEILIN, D., AND HARTREE, E. F. (1951) B&hem. J. 49, 88-104 KEILIN, D., AND HARTREE, E. F. (1955) Biochem. J. 61, 153-

171 SAUNDERS, B. C., HOLMES-SIEDLE, A. G., .IND STBRIC, B. P.

(1964) Perozidase, Butterworths, London YONET.4~1, T., AND ASAKURA. T. (1969) J. Biol. Chem. 244.

4580-4588 LA PLANCHE, L. A., AND ROGERS, M. T. (1964) J. Smer. Chem.

Sot. 86. 337-341 WIBERG, K. B., AND NIGH, W. G. (1965) J. Amer. Chem. Sot.

87, 3849-3855

1, p: 167, W. A‘. Benjamin Inc., N. Y. I - PLAPINGXR. Ii. E. (1959) J. Ora. Chem. 24. 802 GREEN, N. &I. (1963) B&hem. j. 89, 599-609 GORDON, J. A., AND JP~NCKS, W. P. (1963) Biochemistry 2, 47 GORDY, W.: AND STANFORD, S. C. (1941) J. Chem. Phys. 9,204 ARNETT, E. M., AND Wu, C. Y. (1960) J. Amer. Chem. Sot.

82, 49993000 BAKER, A. W., AND SHULGIN, A. T. (1959) J. Amer. Chem.

sot. 81, 1523-1529 GORDON, J. E. (1961) J. Org. Chem. 26, 738 BELL, R. P. (1959) The Proton in Chemistry, p. 161, Cornell

University Press, Ithaca, N. Y. JENCKS, W. P. (1959) Catalysis in Chemistry and Enzymology,

p. 323, McGraw-Hill Co., N. Y. RUPLEY, J. A., BUTLT:R, L., GERRING, &I., JARTDEGEN, F. J.,

AND PECOURO, R. (1967) Proc. h-at. Acad. Sci. U. S. A. 67, 1088

KARUSH, F. (1962) Advan. Zmmun. 2, 1 EISEN, H. N., AND SISICIND, G. W. (1964) Biochemistry 3, 996 WATSON, H. C. (1966) in Hemes and Hemoproteins (CHANCE,

B., ESTABROOIC, R., AND YONF,TANI, T., eds) p. 63, Academic Press, N. Y.

GRUNDNES, J., END KL~BO~, P. (1970) in Chemistry of the Cyano Group (RaPPOPORT, Z., Editor) p. 124, Interscience Publishers, N. Y.

NOBBS, C. L., WATSON, H. C., AND KENDRE~, J. C. (1966) 1Vature 209, 339--341


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Gregory R. SchonbaumNew Complexes of Peroxidases with Hydroxamic Acids, Hydrazides, and Amides

1973, 248:502-511.J. Biol. Chem.

http://www.jbc.org/content/248/2/502Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/248/2/502.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/248/2/502

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;248/2/502&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/2/502

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=248/2/502&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/248/2/502

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/248/2/502.full.html#ref-list-1

http://www.jbc.org/

new complexes of peroxidases with hydroxamic acids ...hydrazides, and benznmide were made in water,...

Documents