1 kinetic and stability properties of p. chrysogenum atp

1

Kinetic and Stability Properties of P. Chrysogenum ATP Sulfurylase Missing the C-

Terminal Regulatory Domain†

Eissa Hanna‡, Kit Fai Ng

§, Ian J. MacRae

‡, Christopher J. Bley

‡,

Andrew J. Fisher§, ‡

and Irwin H. Segel‡, *

Section of Molecular and Cellular Biology and Department of Chemistry

University of California, One Shields Avenue, Davis, CA 95616

Running title: P. chrysogenum ATP Sulfurylase Missing the Allosteric Domain.

Subject area: Enzyme Catalysis/Regulation

Key words: ATP sulfurylase, from P. chrysogenum; sulfurylase; ATP, lacking the allosteric

domain; Allosteric inhibition, of ATP sulfurylase; Regulatory domain, of ATP sulfurylase; PAPS

(3’-phosphoadenosine 5 phosphosulfate), binding to ATP sulfurylase; sulfate activation,

regulation of; selenate, activation by ATP sulfurylase; chromate; arsenate; tungstate; chlorate;

perchlorate; fluorosulfonate.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 12, 2003 as Manuscript M311317200 by guest on A

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ABSTRACT

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to

allosteric inhibition by 3’-phosphoadenosine 5’-phosphosulfate (PAPS). In contrast to the wild

type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was

monomeric and non-cooperative. All kcat values were decreased (the APS synthesis reaction to

17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or

molybdate), the dissociation constant of E·APS, and the monovalent oxyanion dissociation

constants of dead end E·MgATP·oxyanion complexes were all increased. APS release (the k6

step) was rate limiting in the wild type enzyme. Without the C-terminal domain, the composite k5

step (isomerization of the central complex and MgPPi release) became rate limiting. The

cumulative results indicate that beside (a) serving as a receptor for the allosteric inhibitor, the C-

terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits.

Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more

steps following the formation of the binary E·MgATP and E·SO42-

complexes, and preceding the

release of MgPPi is optimized. The more negative entropy of activation of the truncated enzyme

for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective

orientation of MgATP and sulfate at the active site.

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(INTRODUCTION)

Most plants and microorganisms can use inorganic sulfate as their sole source of sulfur.

Because sulfate is nonreactive at cellular temperatures and pH, the anion must first be “activated”

in order to enter the mainstream of metabolism. Activation proceeds in two steps. These are

catalyzed, in order, by the enzymes ATP sulfurylase (MgATP: sulfate adenylyltransferase, EC

2.7.7.4) and APS kinase (MgATP: APS 3´-phosphotransferase, EC 2.7.1.25). The sequential

reactions produce the sulfonucleotides APS1 (adenylylsulfate; adenosine 5’-phosphosulfate) and

PAPS (3’-phopshoadenylylsulfate; 3´-phosphoadenosine 5´-phosphosulfate):

MgATP + SO42- MgPPi + APS (ATP sulfurylase)

MgATP + APS MgADP + PAPS (APS kinase)

ATP sulfurylase from the filamentous fungus, Penicillium chrysogenum, is a

homooligomer composed of six 63.7 kDa subunits (573 residues). PAPS, the APS kinase product,

is an allosteric inhibitor (1,2). This inhibition may be part of a sequential feedback process

considering that PAPS is a major branch point metabolite in filamentous fungi, but not in other

organisms. (PAPS enters into the cysteine biosynthetic pathway and is also used by filamentous

fungi for the formation of choline-O-sulfate, a sulfur storage compound and/or osmoprotectant

(3-6)).

P. chrysogenum ATP sulfurylase is organized as a dimer of triads (7-9). Each subunit

is composed of three structurally distinct globular regions: Residues 1-170 compose a distinct N-

terminal domain. Residues 171-395 compose the central catalytic domain. Several residues that

have been shown to be essential for activity (10,11) are located in this domain. Residues 331–389

form a small subdomain, called Domain III in the yeast structure (12,13). The allosteric site is

located in a C-terminal domain that is very similar to APS kinase in sequence (14) and structure

(15,16). However, this regulatory domain (residues 396 – 573) has no APS kinase activity


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because of modifications to the ATP P-loop (17) and the filling of the ATP binding region with

protein side chain surrogates (e.g., Phe-548, which fills the space that would otherwise be

occupied by the adenine ring of ATP). PAPS is believed to initiate the allosteric transition by

disrupting a salt link between Arg-515 in the C-terminal domain of one subunit and Asp-111 in

the N-terminal domain of a trans-triad subunit (9). In moving from the high-substrate-affinity R

state to the low-substrate-affinity T sate (18-20), the side-chain of Arg-515 moves toward PAPS,

the allosteric domain of each subunit pivots 27° relative to the catalytic and N-terminal domains,

and the hexamer expands slightly in volume. The R to T transition is accompanied by the

movement of a catalytic domain loop (residues 228 – 238, termed the active site switch), which

flips “up” by 17 Å. Rotation about the interface between catalytic-allosteric domains provides the

space for the switch to open. When the switch is in the closed position, Asp-234 interacts with

and presumably modulates the charge on Arg-199 of the active site 1 9 7

QTRN200

sulfate/phosphosulfate motif (8,9). We have suggested that the allosteric effector may not induce

a totally new subunit conformation, but rather, may exploit the existing flexibility of the enzyme.

Small switch movements may be part of the normal catalytic cycle allowing each subunit to act

independently with the “up” switch position corresponding to a low affinity (ligand release)

conformation. A large switch movement in any one subunit may trigger the concerted allosteric

transition.

In order to learn more about the allosteric transition, and particularly, more about the

functional relationship of the of the C-terminal domain to the rest of the protein, we have

examined the properties of recombinant P. chrysogenum ATP sulfurylase missing residues 396-

573. The results indicate that the C-terminal domain does more than just serve as a receptor for

the allosteric inhibitor.


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MATERIALS AND METHODS

Coupling Enzymes and chemicals — Recombinant APS kinase and wild type ATP sulfurylase

from P. chrysogenum were expressed and purified as described earlier (17,21). Yeast ATP

sulfurylase was obtained from Sigma. PAPS was prepared using yeast ATP sulfurylase (Sigma,

A-8957) and fungal APS kinase as described previously (2) and purified by Q-Sepharose

(Pharmacia) chromatography using a 0 – 1 M NaCl gradient in 40 mM Tris-Cl, pH 8.0. The

pooled fractions (50 ml) contained about 2 mM PAPS and 0.3 M NaCl. (PAPS was measured by

the reverse ATP sulfurylase reaction after adding nuclease P1 to convert PAPS to APS.) Most of

the other assay chemicals and coupling enzymes were Sigma products as listed earlier (22).

Protein assays — During purification, the protein concentration was estimated from the A280nm

of the preparation. Specific activities of the final preparation are based on protein concentration

determined with the BCA assay [Pierce Handbook and Catalog, pp 210 - 211, 1989] using bovine

serum albumin as a standard. With the purified enzymes, essentially the same results were

obtained using the relationship [protein]mg/ml = A280nm/E where E = 0.76 for the wild type

enzyme and 0.92 for the truncated enzyme (Biopolymer Calculator at http://

paris.chem.yale.edu/extinct.html). Similar concentrations were obtained from the A280nm and

A235nm values (23) , or the A280nm and A260nm values (24).

Cloning of Truncated ATP Sulfurylase—A DNA encoding residues 1-395 of P. chrysogenum

ATP sulfurylase was generated by PCR using the primers PcATS307 (5’-

TAACTGCAGCATATGGCCAACCTTCACGG-3’ ) and PcATS321 (5 ’ -

AATCTAGATCTTTACTGGGTGGCGCGAGGG-3’), which places a stop codon after residue

395. PCR was carried out with the DNA polymerase Pfu (Stratagene) using a cloned cDNA copy

of the full-length gene as the template. The PCR product was subcloned as a PstI–XbaI fragment


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into the plasmid pBluescript (KS+) and sequenced to ensure no mutations arose during PCR

amplification. The sequenced insert was then cloned as an NdeI-BglII fragment into the Novagen

plasmid pET23a(+) and introduced into E. coli strain BL21(DE3) by electroporation for protein

expression.

Enzyme Expression and Purification—About 0.2 ml of an overnight culture was used to

inoculate two 3-liter Fernbach flasks each containing one liter of LB ampicillin medium. The

cultures were grown aerobically at 37 ˚C until reaching an A600 of 0.8 (about 5-7 hours). Cultures

were subsequently cooled to 15 ˚C and protein expression was induced by the adding IPTG to a

final concentration of 1 mM. After 12-16 h at 15 °C, the cells were harvested by centrifugation at

9,000 -12,000 x g for 20 min. The cells were resuspended in about 40 ml of chilled 40 mM Tris-

Cl buffer, pH 8.0 (standard buffer), containing 1 mM EDTA and lysed in a single pass through a

Watts Fluidair Microfluidizer (model B12-04DJC M3). All subsequent steps were carried out at

4° C.

Cell debris and unbroken cells were removed by centrifugation at 39,000 g for 30 min.

The supernatant fluid was applied to an Affigel Blue column (2.5 x 10 cm) that had been

previously equilibrated with standard buffer. After washing the column for 12 hr at 0.6 ml per

min with the same buffer, the protein was eluted at 2 ml per min with 500 ml of a 0 to 2 M

gradient of NaCl in standard buffer. Ten-ml fractions were collected and those with the highest

A280 nm were pooled (total volume about 70 ml) and dialyzed against standard buffer. The

Affigel Blue fraction was then applied to a Q Sepharose Fastflow column (2.5 x 10 cm)

equilibrated with standard buffer. After washing the column as described above, protein was

eluted at 2 ml per min with a 0 to 1.5 M NaCl gradient in standard buffer. Two ten-ml fractions

containing the highest A280 nm were pooled and dialyzed. When the expression level was very

high and the preparation sufficiently pure after the Affigel Blue column, the enzyme was eluted


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from the Q Sepharose column using a 1 to 2 M NaCl gradient. The final pooled preparation

contained a total of about 50 mg of protein (at 2.4 mg per ml) and was at least 95% pure as

judged by SDS gel electrophoresis.

Enzyme assays — ATP sulfurylase activity was measured by the continuous, coupled

spectrophotometric assays described earlier (22,25,26). These include (a) molybdolysis (coupled

to myokinase, pyruvate kinase, and lactate dehydrogenase), APS synthesis (coupled to APS

kinase, pyruvate kinase, and lactate dehydrogenase, and (c) ATP synthesis (i.e., the reverse

reaction, coupled to hexokinase and glucose-6-phosphate dehydrogenase). All assay mixtures

contained inorganic PPiase. Additionally, APS kinase (ca.1 Unit/ml) was usually included in the

molybdolysis reaction mixture to remove any APS that might be produced from traces of

inorganic sulfate present in the coupling enzymes, buffers, etc. (2). The reaction was usually

started by adding molybdate or sulfate after a 5 to 15 min equilibration period. After another 0.5 -

1 min, ∆A340nm readings were recorded automatically over each 0.1 min interval for the next 2

min. The enzyme concentration was varied to yield a ∆A340nm that was between 0.02 and 0.05

per min as measured on a Perkin-Elmer Lambda 11 spectrophotometer. APS kinase was omitted

when APS was added as an inhibitor. In these experiments, the reaction was started by adding

APS and molybdate simultaneously. Also, the stock MgATP solution was prepared immediately

beforehand from the solid in order to minimize the content of contaminating PPi (27). (PPi would

deplete some of the APS and thus, yield an artificially high Kiq value.) Unless indicated

otherwise, assays were performed in 0.05 M Tris-Cl, pH 8.0 at 30° C. In all cases, activity is

expressed in Units per mg protein where one Unit is equivalent to the formation of 1 µmole of

primary product per minute.


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Data analysis — Initial velocity kinetics of the APS synthesis and molybdolysis reactions were

analyzed by plots of v versus [substrate] at various fixed cosubstrate concentrations. Duplicate

experiments were performed in which the substrate/cosubstrate relationship was reversed. The

data for each series of plots (in the absence of PAPS, etc.) were fitted to the Henri-Michaelis-

Menten equation to obtain Vmax,app and the Km,app for the varied substrate. Replots of Vmax,app

versus the cosubstrate concentration yielded the limiting Vmax and the Km of the cosubstrate. The

same data were analyzed by double reciprocal plots and the appropriate replots. Consequently,

each kinetic constant for the APS synthesis and molybdolysis reactions was determined from two

or three different plots or curve-fits. Activity in the ATP synthesis direction was analyzed by (a)

double reciprocal plots of 1/v versus 1/[PPi] at 500 µM (saturating) APS to obtain Vmax,r and the

Michaelis constant for PPi (KmP) and (b) continuous A340 tracings at 1 mM PPi and 2 µM initial

APS. In the latter, the Michaelis constant for APS (KmQ) was estimated as the concentration of

APS remaining when the tracing velocity was half-maximal. (Vmax was obtained in a separate

experiment at 0.2 mM APS.) The data were also fit to the integrated rate equation for a

unireactant enzyme:

tK

VSS

S SV

m= +−

max maxln

[ ][ ]

[ ] [ ]0 0 [1]

The inhibitory effect of PAPS was determined by fitting the vi/v0 (fractional velocity)

versus [I] data to Eq [2] where vi is the velocity in the presence of inhibitor, v0 is the velocity at

the same substrate concentrations in the absence of inhibitor, Z is the starting value of vi/v0 at [I]

= 0, M is the maximum change in vi/v0, [I] is the inhibitor concentration, nH is the Hill


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coefficient, and K is a constant. (K is equivalent to [I]0.5nH

.) Theoretically, Z = 1.0. If saturating

PAPS drives the velocity to zero, M would also equal 1.0.

vv

ZM I

K Ii

n

n

H

H0= −

+

* [ ]

[ ] [2]

The limiting Ki values for thiosulfate, monovalent oxyanions, and APS and for PAPS

binding to the catalytic site of the truncated enzyme were determined from double reciprocal

plots and slope replots. The Ki for PAPS binding to the truncated enzyme was also estimated

from the [I]0.5,app value of a vi/v0 versus [PAPS] plot at fixed subsaturating [MgATP] and

[MoO42-

]:

KI

AK

BK

A BK K

iapp

ia ib ia mB

=+ + +

[ ]

[ ] [ ] [ ][ ]. ,0 5

1 [3]

DeltaGraph Pro 4.05c was used for all curve fits. Kinetic constants are reported as the

mean determined from multiple experiments (or multiple plots/curve fits). The maximum

variations were generally less than ± 15% of the mean.


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RESULTS

Native and Subunit MW — The truncated enzyme was active and eluted from a Sephacryl S-

100-HR column at a position partially overlapping (but slightly behind) that of fungal APS kinase

(47.4 kDa). SDS gel electrophoresis yielded a subunit size of about 46 kDa. The theoretical

subunit MW is 44 kDa, so it appears that the truncated enzyme is monomeric. Wild type fungal

ATP sulfurylase is a hexamer organized as a dimer of triads in the shape of an flattened ellipsoid

134 Å diam x 73 Å (8). Each triad is stabilized by the head-to-tail interaction of a catalytic

domain of one subunit with the C-terminal domain of the next. In addition, each C-terminal

domain interacts across the triad interface with an N-terminal domain, a catalytic domain, and

another C-terminal domain. Considering the many oligomer stabilizing interactions of the C-

terminal domain, it is not surprising that its absence results in a monomeric enzyme.

Stability of the Truncated Enzyme — Truncated P. chrysogenum ATP sulfurylase is much less

heat stable than the wild type enzyme. At temperatures above 30° C, activity is lost in a first order

fashion, as shown in Fig 1A. To obtain a comparable series of inactivation curves for the wild

type enzyme, a temperature range of 55° to 65° C is required (Fig. 1B). For example, t1/2 for

inactivation of the truncated enzyme at 50 ° C is about 0.3 min while the wild type enzyme is

stable for >2 hr at that temperature. At 45° C, the truncated enzyme has a t1/2 of about 1.5 min.

To obtain the same t1/2 for the wild type enzyme, T must be increased to about 62° C. Ea for

inactivation of the wild type and truncated enzymes are 107 kcal/mole and 62.3 kcal/mole,

respectively (Fig. 1C). Clearly, hexamerization not only provides the means of propagating a

concerted allosteric transition (18-20), but also confers thermal stability. This was not surprising

considering the multiple contacts made by each C-terminal domain of the wild type enzyme as

noted above.


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Sensitivity to Sulfhydryl and Arginine-Targeted Reagents —Preincubation of the wild type

enzyme (15 nM in active sites in 50 mM K-phosphate buffer, pH 8.0, 30° C) with 50 µM DTNB

or 150 µM NEM resulted in a rapid decrease in activity subsequently measured at 50 µM MgATP

and 100 µM MoO42-

(subsaturating substrate levels). The t1/2 values for the two reagents were 20

sec and 45 sec, respectively. This apparent inactivation (which is observed only at subsaturating

substrate levels) is caused by increases in the [S]0.5 values for both substrates concomitant with

the induction of sigmoidal kinetics (28). Under the same preincubation conditions, the truncated

enzyme retained >97% of its activity after 30 min. The results confirm that the effect of SH-

reactive reagents on the wild type enzyme resulted solely from Cys-509 modification. Two other

Cys residues (located in the N-terminal domain at positions 42 and 69) appear to be inaccessible

to DTNB and NEM.

Both forms of the enzyme were irreversibly inactivated by 3 mM phenylglyoxal, an

arginine-targeted reagent (29). (Activity was measured at 5 mM MgATP and 5 mM MoO42-

).

While there are many Arg residues in ATP sulfurylase, the loss of activity must result, at least in

part, from modification of Arg-199 at the active site. (Substrates protect against inactivation

(28)). The t1/2 values were 5 min for both forms of the enzyme indicating no major difference in

the accessibility of essential Arg residues.

pH Profiles— At 1 mM MgATP and 5 mM molybdate, the molybdolysis reaction rates were

nearly constant between pH 6.5 and 9.5 for both the wild type and the truncated enzyme (Fig.

2A). At subsaturating substrate concentrations, the wild type enzyme displayed what appeared to

be a typical “pH optimum” curve, but the response of the truncated enzyme was still essentially

flat (Fig. 2B). Consequently, the usual explanations for the pH effect were inapplicable. That is,

the decrease in activity at lower pH values displayed by the wild type enzyme can not be


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attributed to a reduction in the level of the true substrate, MgATP2-

(which would be minimal

anyway (30,31)). Nor could it result from protonation of essential His residues (10,11), which are

believed to play a role in MgATP binding (32,33). If these causes were relevant, the truncated

enzyme would have behaved the same way. It is more likely that the decrease in activity

exhibited by the wild type enzyme at low pH reflects its transition to the high substrate Km T

state (21), a shift denied to the truncated enzyme. The Scatchard plots shown in Fig. 2C confirm

that the wild type enzyme behaves cooperatively at pH 6.5, but the truncated enzyme displays

normal hyperbolic behavior.

Inhibition by PAPS — At 0.5 mM MgATP and 0.1 mM molybdate, the wild type enzyme

displayed a sigmoidal PAPS inhibition curve with a Hill coefficient (nH) of 2.6 and a [PAPS]0.5

of about 40 µM (Fig. 3). In contrast, neither the truncated P. chrysogenum enzyme nor the yeast

enzyme showed cooperative inhibition. The [PAPS]0.5 values combined with the experimental

substrate concentrations and the appropriate kinetic constants (Eq. [3], Table I, and (14)) yielded

estimates for the limiting Ki values in the region of 60 µM and 180 µM for the truncated P.

chrysogenum and yeast enzymes, respectively. The inhibition of the noncooperative enzymes

almost certainly results from PAPS binding to the APS subsite of the catalytic domain. PAPS is,

after all, a near-perfect structural analog of APS and the crystal structures indicate that only small

changes in the structure of the active site region are needed to accommodate the 3’-phospho

group. (Although it is likely that the PAPS affinity of the wild type enzyme’s active site is closer

to that of the hexameric yeast enzyme than to that of the truncated P. chrysogenum enzyme.) A

more detailed analysis of the inhibition of the truncated enzyme (Fig. 4) yielded a limiting Ki of

71 µM – considerably greater than the Kiq of 0.5 µM for APS binding to its active site (see


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below), but still substantial. The binding of PAPS to the catalytic site, as well as to the allosteric

site of the wild type enzyme, acts to decrease the degree of cooperativity that would otherwise be

observed.

Comparative Activities of the Wild Type and Truncated Enzyme — Table I summarizes the

limiting kinetic constants of wild type and truncated P. chrysogenum ATP sulfurylase at pH 8.0,

30° C. It can be seen that eliminating the C-terminal domain reduces the kcat for molybdolysis

and the reverse (ATP synthesis) reactions by about 40%. In contrast to this moderate effect, the

kcat for the physiological APS synthesis reaction is decreased substantially from 10.8 sec-1

to 1.8

sec-1

. In addition, the Michaelis constants of the truncated enzyme for MgATP and sulfate (or

molybdate) are an order of magnitude greater than those of the wild type enzyme. Truncation has

no major effect on the affinity of the active site for MgATP and sulfate (i.e., Kia and Kib are

essentially unaffected.) The substrate interaction factor, α, defined as KmA/Kia (for MgATP) or

KmB/Kib (for sulfate) is 0.22 for the wild type enzyme but 2.5 for the truncated enzyme. The

difference was equally pronounced for the molybdolysis reaction (0.03 versus 0.24). Because the

kinetic mechanism is not completely rapid equilibrium, the Michaelis constants are not simple

dissociation constants. Consequently, the increase in Km resulting from the loss of the C-terminal

domain can not be attributed solely to a decrease in the affinity of a binary E·S complex for the

cosubstrate, although this could be a factor. A change in downstream rate constants, including

those for catalysis and product release, may also play a role (see later).

The apparent equilibrium constant of the reaction obtained from the Haldane equation

(Table I) differs by a factor of 2.7 for the two enzyme forms. But this is certainly a result of the


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cumulative error introduced when calculating Keq as the product of six experimental kinetic

constants. (Keq should be the same regardless of the enzyme used to catalyze the reaction.)

The kinetics studies described below were performed in to identify, or at least narrow the

choice of the step(s) that were affected by the loss of the C-terminal domain.

Reactivity With Other Inorganic Substrates— ATP sulfurylase is rather non-specific for the

inorganic substrate, accepting a variety of divalent oxyanions (Table II). Sulfate and

fluorophosphate yield stable nucleotides that can be isolated (34,35). Selenate yields APSe which

is unstable but has a lifetime long enough to be captured by APS kinase and phosphorylated to

become PAPSe (36-38). The t1/2 of PAPSe is estimated to be several minutes (38). Tungstate and

chromate, like molybdate, do not produce long-lived stable nucleotide products, but rather,

promote the overall hydrolysis of ATP to AMP plus PPi. Arsenate shows slight activity in the

APS kinase coupled assay, but for the present we can not exclude the possibility that this activity

resulted from contaminating sulfate. (Contamination of the stock Na2HAsO4·7H2O with 0.2%

Na2SO4 by weight would account for the observed activity (38)). As shown in Table II,

truncation results in increased Michaelis constants with almost every divalent oxyanion substrate

indicating that the C-terminal domain affects a step that is common to all of the reactions

catalyzed. The wild type enzyme also displayed low activity with phosphate in the absence of

APS kinase or myokinase. Submillimolar levels of APS and monovalent oxyanions were strong

inhibitors of the reaction, confirming that the activity was that of ATP sulfurylase, and not a

contaminant. (For example, at 25 mM Pi and 2 mM MgATP, the reaction was inhibited 50% by

30 µM FSO3-; data not shown.)


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Product Inhibition — APS was competitive with both substrates and bound more tightly to the

active site of the wild type enzyme than to the truncated enzyme (Table III). Considering that

MgATP alone and sulfate (or molybdate) alone bind to their respective subsites equally well on

both enzyme types, the difference in Kiq suggests a role of the C-terminal domain in shaping the

composite phosphosulfate subsite of the catalytic domain.

Pyrophosphate was noncompetitive with respect to MgATP and sulfate, but the truncated

enzyme yielded double reciprocal plots of 1/v versus 1/[MgATP] at different fixed [MgPPi] that

intersected very close to the vertical axis. Kip,app at 10 mM sulfate was 330 µM. With the wild

type enzyme, the apparent inhibition constants for PPi are in the 0.6 - 3 µM region (39). The

results suggest that E·APS, the enzyme species that normally binds MgPPi in the steady state,

accounts for very minor fraction of the truncated enzyme and that most of the inhibition seen at

ca. Kip,app levels resulted from MgPPi competing with MgATP for free E.

Dead end inhibiton — Inorganic thiosulfate was competitive with molybdate and noncompetitive

with MgATP. The Ki for thiosulfate dissociation from E·S2O32-

was calculated to be 1.8 mM for

the wild type enzyme and 2.2 mM for the truncated species -- not a major difference between the

two enzyme forms. The βKi values for thiosulfate dissociation from E·MgATP· S2O32-

were 0.33

mM and 1.4 mM for the wild type and truncated forms, respectively. Thus the wild type enzyme

shows about the same degree of synergism between MgATP and thiosulfate (β = 0.18) as

between MgATP and sulfate (α = 0.22), although thiosulfate does not enter into a reaction. The

interaction factor for thiosulfate, β, was 0.64, (about 3.5 times poorer) for the truncated enzyme.

Monovalent oxyanions, such as fluorosulfonate and chlorate, were competitive with

molybdate and uncompetitive with respect to MgATP. The βKi for fluorosulfonate dissociation


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from E·MgATP·FSO3-

was 7.4 µM for the wild type enzyme, but 820 µM for the truncated

species –- more than a 100-fold difference (Table III). Because there are no downstream product

release steps with dead end oxyanion inhibitors, the effect of truncation on βKi restricts the role

of the C-terminal domain to a step located between the formation and any subsequent

isomerization of a ternary complex, i.e., somewhere within the sequence EA + I EAI

E’AI.

Except for the near-competitive inhibition by MgPPi, the product and dead end inhibition

patterns were the same as those seen with the wild type enzyme at pH 8.0, 30° C (37,39). So

removal of the C-terminal domain did not alter the kinetic mechanism significantly: MgATP and

sulfate (or molybdate, or thiosulfate) bind randomly to the enzyme, monovalent oxyanions bind

almost exclusively to E·MgATP, and product release in the APS synthesis direction is ordered

with MgPPi dissociating before APS (i.e., the mechanism can be described as steady state random

A-B, ordered P-Q).

The non-reactivity of chlorate and nitrate is understandable. Although the oxygen atoms

of these monovalent oxyanions fit nicely between Gln-197, Arg-199, and Ala-295 (main chain

–NH) of the sulfate subsite, they do not have a fourth oxygen to point toward MgATP. In the case

of fluorosulfonate (FSO3-) and perchlorate, (ClO4

-), the fourth oxygen does not carry a

sufficiently negative charge.

The reason for the inactivity of thiosulfate (SSO32-

) is not immediately obvious. If the

197QXRN

200 motif and Ala-295 preferentially H-bond to the three outer oxygen atoms (8), then

the fourth outer atom that is oriented toward ATP would be the less electronegative sulfur. If, on

the other hand, the outer sulfur atom binds to the main chain –NH of Ala-295, as in the crystal


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17

structure of the yeast enzyme, (13), then another reason must be sought. Perhaps small

differences in bond lengths make a difference. The S–S bond in thiosulfate is 2.1 Å long

compared to 1.7 Å of the S–O bond. This difference may cause the trigonal plane described by

three outer protein-interacting atoms of the inorganic substrate (O, O, and S) to tilt, moving the

remaining negative oxygen away from the α-P of MgATP.

Rate Constants — As shown in the Appendix, the macro kinetic constants of the physiological

reaction can be used to estimate k6 (the rate constant for APS release) and then k5 (a composite

rate constant for MgPPi release and all preceding isomerizations of the central complex). The

calculations indicate that APS release is almost completely rate limiting in the wild type enzyme:

kcat,f = 10.8 sec-1

; k6 = 11.4 sec-1

, k5 = 219 sec-1

. Because kcat,f and k6 are close, the

calculation of k5 probably has considerable error. But it is certainly greater than k6. Also, the

binding of APS to free E is close to being diffusion limited (k-6 is calculated to be 1.8 x 108

M-1

sec-1

). The calculated k-5 was 3.2 x 107 M

-1sec

-1. For the truncated enzyme, kcat,f = 1.8

sec-1

, k6 = 47.5 sec-1

, k5 = 1.9 sec-1

, k-5 = 2.3 x 106 M

-1sec

-1, and k-6 is about 9.3 x 10

7 M

-1

sec-1

. Thus without the C-terminal domain, the overall kcat,f is lower and the earlier composite k5

step becomes rate limiting in the forward direction. (The forward reaction of the truncated

enzyme reduces to a rapid equilibrium condition, as suggested by the altered MgPPi inhibition

data.) The step affected by the C-terminal domain must lie within the sequence EAB

EPQ P + EQ. The only forward reaction step common to this sequence and the

one shown earlier for the effect of the domain on the binding of monovalent oxyanions is

isomerization of a ternary complex.


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Activation energies–– Plots of log Vmax (APS synthesis) versus 1/T (°K-1

) were linear over the

range of 15° to 30° C and yielded Arrhenius activation energies, Ea, of 17.3 and 16.9 kcal per

mole for the wild type and truncated enzymes, respectively. The corresponding ∆H‡ values

(calculated as ∆H‡ = Ea – RT) were 16.7 and 16.3 kcal per mole. ∆G

‡ values calculated from

absolute reaction rate theory (40) were 16.3 and 17.4 kcal per mole for the two enzyme types,

respectively, at 30 ° C. Thus the entropies of activation calculated from ∆S‡ = (∆H

‡- ∆G

‡ )/T

were +1.3 and –3.5 entropy units per mole for the wild type and truncated enzymes, respectively.

In structural terms, the more negative ∆S

‡ for the truncated enzyme could mean that the C-

terminal domain assists in the orientation of the substrates at the active site, a role consistent with

the comparative kinetic properties of the two forms described above.


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DISCUSSION

P. chrysogenum ATP sulfurylase missing the C-terminal allosteric domain is catalytically

active, but it is monomeric and much less stable than the hexameric wild type enzyme. As

expected, the truncated enzyme does not display cooperativity in the presence of PAPS, at low

pH, or after preincubation with an SH-reactive reagent. But in addition, truncation results in (a) a

major reduction in kcat for the physiological reaction and marked increases in (b) the substrate

Michaelis constants, (c) βKi values for monovalent oxyanion inhibitors competitive with sulfate

(d), Kip for MgPPi, and (e) Kiq for APS with (f) little or no change in Kia and Kib values. The

decrease in kcat and the increased Km values for MgATP and sulfate result in part from (g) a

large decrease in the composite k5 step. These kinetic differences indicate that in addition to

providing the binding site for PAPS and stabilizing the hexameric structure, the C-terminal

domain also participates in perfecting the active site. This “activating” effect is focused on a step

that follows the formation of the first central complex (EAB) but precedes the release of MgPPi.

The step may be the alignment of the partially positive α-phosphorous of MgATP with a negative

oxygen of bound oxyanion. When X is sulfate (or molybdate, tungstate, etc.), catalysis then

occurs. But when the first ternary complex contains thiosulfate or chlorate, etc, the structural

change induced by the C-terminal domain just produces a tighter dead end complex. In the

absence of the C terminal domain, the post-EAB reaction between MgATP and sulfate or

molybdate still occurs, but more slowly. If the first ternary complex of the truncated enzyme

contains a non-reactive monovalent oxyanion, the subsequent isomerization is diminished or may

not occur at all. Standard biochemistry texts generally do not credit quaternary structure as

contributing to the function or efficiency of the catalytic site (unless, of course, the site lies at a


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20

subunit interface). However, the literature does contain examples of subunit interactions that help

to perfect a non-interface catalytic site (15,41,42).

A simple scenario for the allosteric transition of the fungal ATP sulfurylase would have

the allosteric domains hold the oligomeric enzyme in a conformation where all subunit catalytic

sites have the same high “proficiency” or overall catalytic competence, i.e., the R state structure.

When the allosteric inhibitor binds, stabilizing linkages are broken and the oligomer undergoes a

transition to the low proficiency T state. The model suggests that the catalytic site of truncated P.

chrysogenum ATP sulfurylase might have T state characteristics. The experimental results are

consistent with this concerted transition model to the extent that the Michaelis constants of the

truncated enzyme for MgATP and sulfate are increased. But the bireactant kinetics of the wild

type enzyme (43) suggests that Kia of the T state is also increased, and that is not observed for the

truncated enzyme.

The importance of the C-terminal domain (or part of it, at least) to structure and function

is further exemplified by yeast ATP sulfurylase (12,13). This enzyme has a hexameric structure

that is very similar to that of the P. chrysogenum enzyme. In fact, the N-terminal and catalytic

domains of the two enzymes (residues 1-395) are 67% identical in sequence and superimpose

with an rms deviation of 0.72 Å for 363 equivalent α-carbons. Yeast and P. chrysogenum

enzymes have very similar kinetic properties (14) except for their responses to PAPS (Fig. 3). At

first glance, there appears to be few similarities between the C-terminal domains of the P.

chrysogenum and yeast ATP sulfurylases. The sequences do not align and the latter is about 50

residues shorter. Nevertheless, the topology of the yeast C-terminal domain reveals that it too

must have evolved from APS kinase (Fig. 5). But the yeast enzyme is not allosterically inhibited

by PAPS. This is not surprising considering that yeast ATP sulfurylase lacks many C-terminal

residues responsible for sulfonucleotide binding. For example, the mobile lid element which


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21

forms half of the binding site for (P)APS in true APS kinase (15) and in the allosteric domain of

P. chrysogenum ATP sulfurylase (9) is completely deleted in the yeast enzyme. The degenerate

C-terminal domain of the yeast enzyme may be a vestigial feature of an ancestral bifunctional

“PAPS synthetase”, parts of which have been retained to stabilize the hexameric structure and to

hold the catalytic domain in a (perpetual) high proficiency conformation2. In this regard, the

structure and kinetic properties of a chimeric enzyme composed of the N-terminal and catalytic

domains of the P. chrysogenum enzyme joined to the C-terminal domain of the yeast enzyme

(and vice-versa) would be informative.

Among ATP sulfurylases of sulfate assimilators, the enzymes from filamentous fungi and

yeast may be maximally optimized for the APS synthesis direction. These hexameric enzymes

have the highest APS synthesis/ATP synthesis kcat ratio (ca. 0.14) of the several ATP

sulfurylases that we have kinetically characterized so far (22,25,34,38), and the Ki and Km values

for MgATP and SO42-

are in the likely intracellular concentration range (ca. millimolar). The C-

terminal domain may be the agent responsible for the extra “tailoring” of the active site. Of

course, optimization must remain under the constraint of the Haldane equation, a relationship that

relates the kinetic constants of the enzyme to the equilibrium constant of the reaction (see Table

I). If evolutionary pressure operated to maximize the forward/reverse kcat ratio and at the same

time, insure a substantial fraction of Vmax,f at cellular levels of ATP and SO42-

, then in the face

of the extremely small (and unalterable) Keq for the APS synthesis direction, only Kiq and/or

KmP would be available for adjustment -- which seems to be the case. That is, the compensation

shows up as much higher affinities for the physiological reaction products (particularly APS) than

for the substrates (44), a seemingly contradictory feature. But in vivo, strong product inhibition by


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APS would not be an obstacle because the next enzyme in the sulfate activation sequence (APS

kinase) has a high affinity for APS (17). Consequently, under normal physiological conditions,

APS would not accumulate to high levels. In contrast to the wild type enzyme, the kcat ratio of

the truncated enzyme is reduced to about 0.05 and the substrate Km values are increased by an

order of magnitude.

X-ray crystallographic studies on the truncated enzyme are in progress. These may reveal

the structural differences in the catalytic domain that are caused by the absence of the C-terminal

regulatory domain.


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APPENDIX: Kinetic constants of ATP Sulfurylase.

The kinetic mechanism of fungal ATP sulfurylase at pH 8 and 30° can be described as

random A-B, ordered P-Q sequence. The reaction scheme is shown below. Positive and negative

rate constants correspond to the “forward” and “reverse” directions, respectively.

A velocity equation that is first degree with respect to substrate concentrations has been derived

assuming that E, A, and B are at equilibrium with the EA and EB complexes (37). For this

mechanism, the rate constant compositions of the limiting macro kinetic constants are as follows:

kk k

k kcat f, =+( )5 6

5 6

, k k kcat r, ( )= +− −2 4 ,

Kkk

Kkkia ib= =− −1

1

3

3, ,

Kk k k k k k

k k k k k k k kmA =+ +( )

+( ) +( )− − −

− −

1 3 6 2 4 5

5 6 1 2 3 1 3 4,

A = MgATP

B = SO42-

P = MgPPiQ = APS

E

EA

EB

A

AB

B

P Qk

1k2 k-2k-1

k3 k4k-3 k-4

k5 k6k-5 k-6

(EAB EPQ) EEQ


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Kk k k k k k

k k k k k k k kmB =+ +( )

+( ) +( )− − −

− −

1 3 6 2 4 5

5 6 1 2 3 1 3 4,

Kkkiq =−

6

6, K

k kkmQ =+− −

−

( )2 4

6, K

k k kkmP =

+ +− −

−

( )2 4 5

5,

Thus k6 can be calculated from:

kk K

Kcat r iq

mQ6 = ,

Then k5 can be obtained from 1 1 1

5 6k k kcat f= −

, or k

k k

k kcat f

cat f5

6

6=

−,

,( ).

And k-5 can be calculated from kk k

Kcat r

mP− =

+5

5, .

k-6 can be calculated from Kiq or from:

kk

Kcat r

mQ− =6

,

k5 calculated as shown above is not just the rate constant for MgPPi dissociation, but

rather, a composite constant composed of the true koff for MgPPi and the rate constants for

isomerization of the central complex. Similarly, k-5 is composed of kon for MgPPi addition to

E·APS and isomerizations of the resulting EPQ complex. In the above formulation, k2, k-2, k4,

and k-4 are also composite constants.


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FOOTNOTES

† The research described in this report was supported by NSF Research Grant MCB 9904003 to I.

H. S. and A. J. F. and by facilities of the W. M. Keck Foundation Center for Structural Biology at

the University of California, Davis.

‡ Section of Molecular and Cellular Biology. E. H., and C.J.B. are Undergraduate Honors

Research Students.

§ Department of Chemistry; K. F. N is a recipient of a Pfizer Summer Undergraduate Research

Fellowship.

* Corresponding author. Phone: (530) 752-3193. E-mail: [email protected].

1Abbreviations: APS, adenosine 5’-phosphosulfate (adenylylsulfate); Ap5A, diadenosine

pentaphosphate; PAPS, 3’-phosphoadenosine 5’-phosphosulfate (adenylylsulfate 3’-phosphate);

MgATP, MgPPi, MgADP, magnesium complexes of the corresponding substrates or products;

PPi, inorganic pyrophosphate, PPiase, inorganic pyrophosphatase; Tris, tris-

hydroxymethylaminomethane; MES, (2-N-morpholino)ethanesulfonic acid; EPPS, N-2-

Hydroxyethylpiperazine-N’-3 propanesulfonic acid; DTNB, 5,5’-dithiobis-(2-nitrobenzoate);

NEM, N-ethylmaleimide; Ea, Arrhenius activation energy; nH, Hill coefficient.

2 The classical “concerted transition” or “symmetry” model for cooperative enzymes considered

only unireactant enzymes (18-20). An extension of the model to multireactant enzymes

introduces additional features. For example, positive cooperativity would be observed with a

bireactant enzyme even if the T and R states have identical affinities for substrates A and B (in

forming the binary EA and EB complexes) as long as the R state has a greater degree of substrate

binding synergism (21,45). In this case, the higher affinity of the R state refers only to the


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formation of the ternary EAB complexes. Furthermore, a Michaelis constant might be composed

of more than the kon and koff rate constants. For these reasons, the R and T states of multireactant

cooperative enzymes are best referred to in terms of their overall catalytic competencies, or

effectiveness, or proficiencies, rather than in the terms of their “affinities”.


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Tab

le I

.

Kin

etic

Con

stan

ts o

f P

. chr

ysog

enum

AT

P S

ulf

ury

lase

a

Con

stan

t

Des

crip

tion

V

alue

Wil

d T

ype

(MW

= 6

3.7

kD

a)

Tru

nca

ted

(MW

= 4

4.4

kD

a)A

PS

Synt

hesi

s

Vm

ax

Max

imal

vel

ocit

y of

APS

syn

thes

is

1

0.2

Uni

ts x

mg

pro

tein

-1

2.5

Uni

ts x

mg

pro

tein

-1

k cat

cat

alyt

ic r

ate

cons

tant

10.

8 se

c-1

1

.8 s

ec-1

Kia

E·M

gAT

P d

isso

ciat

ion

cons

tant

0.

9 m

M

1

.1 m

M

Km

BM

icha

elis

con

stan

t for

SO

42- a

t

0.

29 m

M

3

.6 m

M

s

atur

atin

g M

gAT

P

Kib

E·S

O42-

dis

soci

atio

n co

nsta

nt

1.4

mM

1.4

mM

Km

A M

icha

elis

con

stan

t for

MgA

TP

at

0.2

1 m

M

2

.6 m

Msa

tura

ting

SO

42-

k 5

Rat

e co

nsta

nt fo

r ca

taly

sis

and

/or

MgP

P i

219

sec

-1

1

.9 s

ec-1

rel

ease

k 6R

ate

cons

tant

for

APS

rel

ease

11

.4 s

ec-1

47

.5 s

ec-1

Mol

ybdo

lysi

sV

max

M

axim

al v

eloc

ity

of m

olyb

dol

ysis

22.8

Uni

ts x

mg

prot

ein-1

18.5

Uni

ts x

mg

prot

ein-1


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31

k cat

c

atal

ytic

rat

e co

nsta

nt

24.

4 se

c-1

13

.7 s

ec-1

Kia’

E

·MgA

TP

dis

soci

atio

n co

nsta

nt

0.9

mM

1.1

mM

Km

B’

M

icha

elis

con

stan

t for

MoO

42- a

t

0

.076

mM

0.53

mM

s

atur

atin

g M

gAT

P

Kib’

E

· MoO

42- d

isso

ciat

ion

cons

tant

2.5

mM

2.2

mM

Km

A’

Mic

hael

is c

onst

ant f

or M

gAT

P at

0.0

27 m

M

0.

27 m

Msa

tura

ting

MoO

42-

AT

P S

ynth

esis

Vm

ax

Max

imal

vel

ocit

y of

AT

P sy

nthe

sis

6

9 U

nits

x m

g pr

otei

n-1

6

3 U

nits

x m

g pr

otei

n-1

k cat

cat

alyt

ic r

ate

cons

tant

73.

3 se

c-1

46.

6 se

c-1

Kiq

E·A

PS d

isso

ciat

ion

cons

tant

0

.062

µM

0

.51

µM

Km

PM

icha

elis

con

stan

t for

PP i

at s

atur

atin

g A

PS

9.2

µM

25

µM

Km

QM

icha

elis

con

stan

t for

APS

at s

atur

atin

g P

Pi

0.4

µM

0.

5 µ

M

Keq

Equ

ilibr

ium

con

stan

t for

the

APS

syn

thes

is r

eact

ion

calc

ulat

ed fr

om th

e

Hal

dan

e eq

uati

on:

3

.2 1

0-7

1.2 x

10-7

____

____

____

____

____

____

____

____

____

____

____

____

____

__a C

onst

ants

wer

e d

eter

min

ed in

Tri

s-C

l, p

H 8

.0, 3

0° C

. All

solu

tion

s co

ntai

ned

5 m

M e

xces

s M

gCl 2

ove

r to

tal A

TP

or P

P i.


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32

Table IIReactivity of P. chrysogenum ATP Sulfurylase with Different Inorganic Substrates

a

___________________________________________________________________________ Substrate kcat,app

b KmA,app

c KmB,app

b

(sec-1

) (mM) (mM) wt trunc wt trunc wt trunc SO4

2- 10.2 1.3 0.24 1.3 0.3 5.3

(APS kinase)

FPO32-

2.3 0.7 0.09 5.2 0.11 4.6

(APS kinase)

SeO42-

2.1 1.4 0.55 1.8 0.06 3.2 (APS kinase)

SeO42- 1.0 0.9 0.59 2.4 0.07 3.2

(myokinase)

HPO4

2- 0.4 –

d 3 –

d 18 –

d

(neither)

HAsO42-

11.5 0.11 0.98 1.9 8.0 5.8 (myokinase)

HAsO42-

0.5 0.08 1.25 9.3 3.5 13.5 (APS kinase)

MoO42 23.9 16.8 0.01 0.1 0.1 0.6

(myokinase)

WO42-

26.6 14.1 0.07 1.0 0.2 2.0 (myokinase)

CrO4

2- 3.4 2.0 0.01 0.6 0.006 0.08

(myokinase)

________________________________________________________________________________________________________

a The reactions were carried out in Tris-Cl, pH 8.0, 30° C. The enzyme to which the ATP sulfurylase

reaction was coupled is shown in parentheses below the substrate. Ap5A (135 µM) was included in assays

of the selenate-dependent and arsenate-dependent reactions coupled to APS kinase.bVmax,app and KmB,app were determined obtained by extrapolating the 1/v versus 1/[oxyanion] double

reciprocal plot at 5 mM MgATP. kcat,app was calculated from Vmax,app. KmB,app for Pi is more appropriately

indicated as [Pi]0.5 because the primary velocity curve appeared to be slightly sigmoidal.

c KmA,app was determined from a plot of 1/v versus 1/[MgATP] at 10 mM oxyanion substrate except for

chromate, phosphate, and arsenate, which were maintained at 0.3 mM, 20 mM, and 20 mM, in order.d The truncated enzyme did not have measurable activity with Pi as the inorganic substrate.


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33

Table IIIInhibitors of ATP Sulfurylase

a

______________________________________________________________________________

Inhibitor Type of Inhibitionb Limiting Inhibition Constants

c

Varied Substrate Wild type Truncated

MgATP MoO42-

FSO3-

UC C βKi = 7.4 µM βKi = 820 µM

ClO4-

UC C βKi = 7.9 µM βKi = 617 µM

ClO3-

UC C βKi = 21 µM βKi = 1.1 mM

NO3-

UC C βKi = 69 µM βKI = 3.0 mM

S2O32-

NC C Ki = 1.8 mM Ki = 2.2 mM

βKi = 0.33 mM βKi = 1.4 mM

APS C C Kiq = 62 nM Kiq = 510 nM

PPi NC NC Kipd = 0.6 µM Kip,app = 330 µM

K’ipd = 3 µM

PAPS Ce

Ce -- Ki = 71 µM

______________________________________________________________________________aAll constants were determined at 30° C, pH 8.0 at which the wild type enzyme displays normal

hyperbolic kinetics.

b C = competitive; UC = uncompetitive; NC = noncomptitive (or mixed-type).

c Ki is the dissociation constant of the E·I complex; βKi is the I dissociation constant of the

E·MgATP·Inhibitor complex. For the monovalent oxyanion inhibition studies, [MoO42-

] was

maintained at KmB when [MgATP] was varied; [MgATP] was maintained at Kia when [MoO42-

]was varied. The limiting constants were obtained from appropriate slope and/or intercept replotsas described earlier (20,22).

d Wild type values were taken from (39) where the constants were determined by the average

velocity method (in the absence of PPiase) using SO42-

as the inorganic substrate. Kip is


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34

equivalent to the PPi inhibition constant at saturating SO42-

and zero MgATP. K’ip is equivalent

to the inhibition constant when both substrates are saturating. Kip,app for the truncated enzyme isthe inhibition constant obtained from the slope replot of the 1/v versus 1/[MgATP] plots at 10mM SO4

2-.

e For the truncated enzyme only. PAPS induces sigmoidal kinetics with the wild type enzyme.

Saturating MgATP overcomes the inhibition and drives the enzyme to the R state which yieldshyperbolic kinetics. Saturating MoO4

2- (at subsaturating MgATP) will not drive the enzyme

completely to the R state.


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35

Legends to Figures

Fig. 1. Thermal stability of the truncated and wild type P. chrysogenum enzymes.

Truncated P. chrysogenum ATP sulfurylase was preincubated at about 0.5 mg/ml in 0.05 M Na-

EPPS buffer, pH 8.0 at the indicated temperatures. Periodically, a sample was removed and its

activity was measured at 30° C, 5 mM MgATP and 10 mM molybdate. A. Inactivation curves for

the truncated enzyme (semi-log plots). For clarity, data obtained at 48° C and 51° C are not

shown. B. Effect of temperature on the first order rate constant for inactivation. C. Arrhenius

plots. The Ea values were 107 kcal/mole for the wild type enzyme and 62.3 kcal/mole for the

truncated enzyme.

Fig. 2. Effect of pH on molybdolysis activity. Reaction rates were measured at 1 mM MgATP

and 5 mM molybdate (Panel A) or 0.05 mM of both substrates (Panel B). The buffers were

prepared by mixing 0.05 M Na-MES, pH 6.5 with 0.05 Tris, free base, to the desired pH. Panel C

shows the Scatchard plot of the original v versus [MoO42-

] data obtained at pH 6.5 and 0.25 mM

MgATP. The nH values were 2.1 for the wild type enzyme and 1.08 for the truncated enzyme.

Fig. 3. Inhibition by PAPS. Rates were measured under standard assay conditions at 0.5 mM

MgATP, 0.1 mM molybdate, and the indicated concentrations of PAPS.

Fig. 4. Competitive inhibition of the truncated P. chrysogenum enzyme by PAPS. (a) 1/v

versus 1/[MgATP] at 0.5 mM MoO42-

and the indicated concentrations of PAPS. The slope

replot gives –Ki,app as the horizontal-axis intercept where Ki,app = Ki(1 + [MoO42-

]/Kib). (b) 1/v

versus 1/[MoO42-

] at 0.25 mM MgATP. The slope replot gives –Ki,app as the horizontal-axis

intercept where Ki,app = Ki(1 + [MgATP]/Kia).

Fig. 5. Superposition of P. chrysogenum (blue) and S. cereviseae (yellow) ATP sulfurylases.

Protein coordinates of the P. chrysogenum (8,9) and S. cereviseae (12,13) enzymes are available

in the Protein Data Bank (IDs 1M8P and 1JEC respectively). Left: complete subunits. Right: C-

terminal domains of the two enzymes. The location of the allosteric site is shown by the stick

model of bound PAPS.


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1/T (°K-1)

BB

B

B

B

B

B

J JJ

J

J

J

J

0

1.0

2.0

3.0

30 40 50 60 70

k

(min-1)

T (oC)

Wild TypeTruncated

Rem

aini

ng A

ctiv

ity (

%)

B B B B B B B B

J

JJ

JJ

JJ

J

H

H

H

H

H

H

H

F

F

F

F

F

F

F

F

Ñ

Ñ

Ñ

Ñ

1

10

100

0 2 4 6 8 10 12 14 16 18

Time (min)

B

B

B

B

B

B

B

J

J

J

J

J

JJ

-2.0

-1.5

-1.0

-0.5

0.0

0.5

0.0029 0.0030 0.0031 0.0032 0.0033

log k

Wild Type

Truncated

35° C

40° C

42.5° C

45° C

50° C

A

B

C

Fig. 1


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J J J J J J JJ

Ñ ÑÑ Ñ Ñ Ñ

Ñ

Ñ

0

5

10

15

20

6.5 7.0 7.5 8.0 8.5 9.0 9.5

v

(Uni

ts/m

g pr

otei

n)

pH

Truncated

Wild Type

1 mM MgATP

5 mM MoO42-

Ñ ÑÑ

Ñ

Ñ

Ñ

Ñ

Ñ

Ñ Ñ Ñ

Ñ

Ñ

J J J J J J J J J J J J J0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH

v

(Uni

ts/m

g pr

otei

n)

Truncated

Wild Type

0.05 mM MgATP

0.05 mM MoO42-

A

B

ÑÑ

Ñ

Ñ

ÑÑ

Ñ

Ñ

Ñ

JJJ

JJ

J

0

2

4

6

8

10

0 2 4 6 8 10 12 14

v (Units/mg protein)

Wild Type

Truncated

0.25 mM MgATP

pH 6.5

C

v

[MoO

42-]

Fig.2


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BBB

B

B

B

B

B

BB B B

J

J

J

J

J

J

H

H

HH

H H

0.0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100 120

[PAPS] (µM)

MgATP = 0.5 mMMoO4 = 0.1 mM

viv0

truncated P. chrysogenum

wild type S. cereviseae

wild type P. chrysogenum

Fig. 3


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BB

B

B

B

J

J

J

J

J

H

H

H

H

H

F

F

F

F

F

0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2 0.4 0.6 0.8 1.0 1.2 1.4

B

B

B

B

0

0.2

0.4

0.6

0.8

-100 -50 0 50 100 150

1/ MgATP (mM-1)

[MoO4 2-] = 0.5 mM

Slo

pe

[PAPS] (µM)

B

B

B

B

B

J

J

J

J

J

H

H

H

H

H

F

F

F

F

F

0

0.5

1.0

1.5

2.0

0.00.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

B

B

B

B

0

0.2

0.4

0.6

0.8

1.0

1.2

-100 -50 0 50 100 150

[PAPS] (µM)

Slo

pe

1/v

(m

g pr

otei

n/U

nit)

[PAPS] (µM)

150

100

50

0

1/[MoO42-] (mM-1)

[MgATP] = 0.25 mM

1/v

(m

g pr

otei

n/U

nit)

150

50

100

0

[PAPS] (µM)

Fig. 4


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P. c

hrys

ogen

um

S. c

erev

isea

e

P. c

hrys

ogen

um

Sub

unit

C-T

erm

inal

dom

ainS. c

erev

isea

e

Fig

. 5


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SegelEissa Hanna, Kit Fai Ng, Ian J. MacRae, Christopher J. Bley, Andrew J. Fisher and Irwin H.

C-terminal regulatory domainKinetic and stability properties of P. Chrysogenum ATP sulfurylase missing the

published online November 12, 2003J. Biol. Chem.

10.1074/jbc.M311317200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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1 kinetic and stability properties of p. chrysogenum atp

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