Binding of Purine Nucleotides to Two Regulatory Sites Results in

THE Jurns.il. ut BI~I .OW~AI . CtimllsTn\ 0 1994 by The American Society for Blochemlstry and Molecular Biology, Inc.

Vol. 269. No Issue of March 4, pp . 6784-6789, 1994 Printed in U.S.A.

Binding of Purine Nucleotides to Two Regulatory Sites Results in Synergistic Feedback Inhibition of Glutamine 5-Phosphoribosylpyrophosphate Amidotransferase*

(Received for publication, July 6 , 1993, and in revised form, October 26, 1993)

Gaochao ZhouSQ, Janet L. Smithl, and Howard ZalkinS From the Departments of $Biochemistry and Wiological Sciences, Purdue University, West Lafayette, Indiana 47907

Glutamine 5-phosphoribosylpyrophosphate amidotransferase from Escherichia coli is subject to synergistic feedback regulation by adenine and guanine nucleotides. Inhibition assays and equilibrium binding measurements have established that synergistic inhibition by AMP and GMP results from synergistic binding to two sitedenzyme subunit in the homotetramer. Although each nucleotide can bind to both sites, analyses of the wild type and mutant enzymes indicate that binding of GMP to an A (allosteric) site and AMP to a proximal C (catalytic) site are necessary for synergistic inhibition. K326Q and P410W amino acid replacements result in decreased binding affinity for GMP and AMP and lead to corresponding reductions in feedback inhibition. The K326Q A site mutation results not only in decreased affinity of GMP for the mutantAsite but also has an adverse effect on AMP affinity for the C site. Similarly, the P410W C site mutation has a detrimental effect on binding of AMP to the mutant C site and also on affinity of GMP to the A site. The fact that a mutation in one site affects binding of nucleotides to both sites provides further evidence for synergistic binding of nucleotides.

Glutamine 5-phosphoribosylpyrophosphate (PRPP)’ amidotransferase catalyzes the first committed reaction of de novo purine nucleotide synthesis: glutamine + PRPP + phosphori- bosylamine + glutamate + PPi. The synthesis of purine nucleotides is controlled in bacteria by gene regulation and by end product inhibition of the amidotransferase (Zalkin and Dixon, 1992). Genes encoding the Escherichia coli (Tso et al . , 1982) and Bacillus subtilis (Makaroff et al . , 1983) amidotransferase have been cloned and the enzymes purified to homogeneity (Messen- ger and Zalkin, 1979; Wong et al . , 1981). Although the B. subtilis enzyme contains a [4Fe-4S] cluster not found in the E. coli amidotransferase, the two enzymes share -40% amino acid sequence identity and are presumably homologous (Makaroff et al., 1983). The x-ray structure of the B. subtilis amidotrtnsfer- ase containing bound nucleotides has been solved at 3.0-A reso- lution.2 Residues implicated in substrate binding and catalysis are in close proximity to bound nucleotides in the structural model. As a consequence of the extensive conservation of resi-

Grants GM24658 (to H. Z.) and DK42303 (to J. L. S.). This is Journal * This research was supported by United States Public Health Service

Paper 14049 from the Purdue University Agricultural Experiment Sta- tion. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 6 Present address: Dept. of Biological Chemistry, University of Michi-

gan Medical School, Ann Arbor, M i 48109-0606. The abbreviation used is: PRPP, 5-phosphoribosylpyrophosphate. J. L. Smith, E. J. Zaluzec, J-P. Wery, L. Niu, R. L. Switzer, H. Zalkin,

and Y. Satow, submitted for publication.

dues in the catalytic and nucleotide-binding sites, we assume structural similarity for these sites in the homologous E. coli amidotransferase.

The availability of detailed structural information provides the opportunity to investigate the mechanism for feedback regulation of the amidotransferase by purine nucleotides. The main characteristics of the feedback regulation of the E. coli enzyme (Messenger and Zalkin, 1979) are as follows, (i) Of the adenine and guanine nucleotides, the 5’-monophosphates are most inhibitory with the effectiveness of GMP being greater than that of AMP. (ii) Inhibition by AMP plus GMP is synergistic. (iii) Inhibition by AMP and GMP is competitive with the substrate PRPP. (iv) In the absence of inhibitor, saturation by PRPP is hyperbolic. PRPP saturation remains hyperbolic with added AMP but is sigmoidal in the presence of GMP. (v) AMP and GMP exhibit cooperativity for inhibition with Hill coefi- cients of 2.0-4.6, The enzyme is a tetramer of identical subunits. These properties can be accommodated by a model in which there are two nucleotide regulatory siteslsubunit with communication between bound AMP and GMP.

The nucleotide regulatory sites in the E. coli amidotransferase were recently probed using techniques of affinity labeling and site-directed mutagenesis (Zhou et al . , 1993). 5‘-p-Fluoro- sulfonylbenzoyladenosine and 8-azidoadenosine-5’-monophos- phate specifically inactivated the amidotransferase by covalent attachment to amino acid residues implicated in nucleotide binding and catalysis. The results were interpreted in terms of a model in which two nucleotide sites are in close proximity to residues important for catalysis. Amino acid residues required for inhibition by GMP were identified by affinity labeling and mutagenesis, but the second site required for inhibition by AMP was not characterized.

The three-dimensional structure of the B. subtilis enzyme is entirely consistent with the two-site model for nucleotide inhibition.2 Two nucleotides are boundsubunit in the crystalline B. subtilis enzyme: one in the presumed catalytic site (C site) and one in a presumed allosteric site (A site). Furthermore, these sites are adjacent so that binding to one site might easily affect binding to the other. Direct visualization of the two sites has led to expanded studies of regulation by nucleotides.

We now report the results of inhibition and equilibrium binding experiments which define the two nucleotide regulatory sites in the E, coli amidotransferase. The results indicate that each nucleotide can bind to both sites. Inhibition by GMP requires binding to the A site whereas inhibition by AMP requires binding to the C site. Synergistic inhibition by AMP plus GMP results from synergistic binding.

EXPERIMENTAL PROCEDURES

scribed (Zhou et al., 1993). A P410W C site mutant and K326QiP410W Enzymes-The K326Q amidotransferase A site mutant has been de-

double mutant were constructed by site-directed mutagenesis (Kunkel et al., 1987) using plasmid pGZ13 (Zhou et al., 1993). Plasmid pGZl3

6784

Glutamine PRPP Amidotransferase Nucleotide Regulatory Sites 6785

contains E. coli purF in phagemid pTU19U (Zhou et al., 1993). Muta- tions were verified by nucleotide sequencing (Sanger et al., 1977). Plas- mid pGZ14 (Zhou et al., 1993) was used for production of the wild type amidotransferase. The P410W replacement, based on the x-ray structure of the enzyme-nucleotide complex, was designed to obstruct the C site with a bulky amino acid side chain.

The wild type and mutant amidotransferases were overproduced in "X358 (purF r e d ) and purified to approximately 90% homogeneity based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (Mei and Zalkin, 1989). Overproduction and puri- fication of the three mutant enzymes were similar to the wild type. The activity with glutamine of the wild type and mutant enzymes was similar as was the activity with NH,.

Enzyme Assay-PRPP-dependent glutaminase activity (Messenger and Zalkin, 1979) was used to assay nucleotide inhibition. Each reaction contained 1 rn PWP, 5 m~ glutamine, 10 or 20 m~ MgCl,, varied concentrations ofAMP or GMP for feedback inhibition, 50 m~ Tris-HC1, pH 7.5, and approximately 700 ng of enzyme in a volume of 0.5 ml. Enzyme activity was similar at either MgClz concentration. However, the enzyme was slightly more sensitive to inhibition by nucleotide at the higher MgCl, concentration. The incubation for 10 min at 37 "C was followed by a glutamate dehydrogenase assay to determine glutamate production (Messenger and Zalkin, 1979). NH3-dependent synthase activity was assayed by a coupling reaction using glycinamide ribonucleotide synthetase (Schendel et al., 1988). A 0.04-ml mixture contained 3 m~ PRPP, 150 m~ NH,Cl, pH 8.0,2.5 rn ATP, 2 rn P4Clglycine (1800 counts/midnmol), 10 m~ MgOAc, 50 m~ Tris-HC1, pH 8.0, and 100 ng of enzyme. Incubation was for 8 min a t 37 "C. Purified glycinamide ribonucleotide synthetase (Shen et al., 1990) was the generous gift of J. Stubbe, Massachusetts Institute of Technology, Cambridge, MA.

Equilibrium Dialysis-The equilibrium dialysis was conducted in 500-pl chambers containing a total volume of 400 pl. A 200-1.11 mixture of 200 m~ Tris-HC1, pH 7.5, 20 m~ MgC12, 70 IIM [8-3H1GMP (0.2 pCi), or 56 IIM [2,8-3H]AMP (0.2 pCi), and a concentration of AMP or GMP from 1 to 1800 p was loaded into one 250-pl well of the chamber. The other well contained 200 pl of enzyme (from 65 to 200 p) in 10 m~ Tris-HC1, pH 7.5. In some experiments, an unlabeled competitor purine nucleotide was included. Radioactive purine nucleotides were pur- chased from Moravek Biochemicals, Inc. A 12-14-kDa cutoff dialysis membrane separated the two wells. The membrane was soaked over- night at 4 "C in 0.1 M EDTA, pH 8.0, rinsed in deionized water, and stored in a buffer containing 100 mM Tris-HC1, pH 7.5, 10 rn MgCl,, and 0.02% sodium azide. Dialysis was for 20 h in a rotating apparatus. Samples of 90-150 pl were counted for radioactivity. Binding data were plotted in two ways: (i) log fractional saturation (Y) uersus free nucleotide and (ii) Scatchard plot (Scatchard, 1949). Data for ligand binding were fit by non-linear regression to the Scatchard equation for one or two sites using Enzfitter (Leatherbarrow, 1987). The number of binding sites, n, and the Kd were determined from the Scatchard plot.

RESULTS Nucleotide Inhibition-E. coli glutamine PRPP amidotrans-

ferase is subject to synergistic inhibition by AMP + GMP (Mes- senger and Zalkin, 1979). The results of affinity labeling by nucleotide analogs (Zhou et al., 1993) and the structural model of the enzyme-nucleotide complex2 indicate two nucleotide sitedsubunit. One nucleotide site has been designated allosteric (A site), the other catalytic (C site). Nucleotide inhibition of mutant and wild type enzymes helps define the binding speci- ficity ofAMP and GMP for the two sites. Three mutant enzymes were analyzed: K326Q (A site) previously shown to be insensitive to inhibition by GMP (Zhou et al., 19931, P410W (C site), and K326Q/P410W double mutant. Inhibition curves for the wild type and three mutants are shown in Fig. 1, A and B. In agreement with previous results (Zhou et al., 1993), GMP was the better inhibitor. The K326Q enzyme was insensitive to inhibition by GMP but retained inhibition by AMP as seen earlier (Zhou et al., 1993). We therefore infer that this mutation interferes with binding of GMP to the A site with consequent loss of inhibition. Binding ofAMP to the C site could account for the residual inhibition by AMP. The P410W C site mutation reduced the capacity for inhibition by AMP perhaps as a result of reduced affinity ofAMP for the C site, but inhibition by GMP was essentially normal. In the double mutant, binding of both

A I2O-

\

0 0 2 4 6 8 10

AMP, mM

B 120

K326QIP410W

1 \P410W

W.T. \

-I 1: E

0 1 2 3 4

GMP, mM FIG. 1. Feedback inhibition of wild type and mutant amido-

transferase by AMP (panel A) and GMP (panel B). Enzyme activity was determined as described under "Experimental Procedures."

nucleotides was evidently abolished resulting in loss of inhibition. These results support a model in which binding of GMP to the A site and AMP to the C site is required for inhibition by the nucleotides (Zhou et al., 1993).

Inhibition by nucleotide mixtures is shown in Table I. Nucleotide concentrations were chosen to emphasize synergistic inhibition of the wild type enzyme by AMP and GMP. Based on the inhibition by 2 m~ AMP or by 0.8 m~ GMP shown in Table I, the wild type enzyme should retain 55% activity in the presence of 2 m~ AMP + 0.8 mM GMP if each inhibitor functions independently. However, these concentrations of nucleotides completely abolished activity. Even at a reduced concentration of AMP + GMP the observed inhibition was greater than that expected for additive inhibition by 2 mM AMP + 0.8 mM GMP. These results emphasize that the two purine nucleotides exert synergistic inhibition.

In the K326Q A site mutant, defective binding of GMP to the mutant A site led to reduced synergistic inhibition, although synergism was not abolished. The results of binding experiments presented below suggest that binding of AMP to the C

6786 Glutamine PRPP Amidotransferase Nucleotide Regulatory Sites

P410W

TABLE I Synergistic inhibition Of QmidOtTUnSferQSeS by AMP plus GMP

der “Experimental Procedures.” The PRPP concentration was 1 mM and PRPP-dependent glutaminase activity was assayed as described un-

MgC1, concentration was 10 m ~ . The calculated residual activity given in parentheses is the product of the activities obtained in the reactions with each inhibitor. Thus the calculated residual activity represents the resulting activity if each inhibitor acts independently.

Enzyme Nucleotide Conc. Activity

r n M B Wild type AMP 2 17

GMP 0.8 12 AMP + GMP AMP + GMP

2 + 0.8 0 (55 ) 1 + 0.4 20

K326Q AMP 2 84 GMP 0.8 98 AMP + GMP AMP + GMP

2 + 0.8 69 (81) 1 + 0.4 88

AMP GMP

2 96

AMP + GMP 0.8 88 2 + 0.8 0 (85)

AMP + GMP 1 + 0.4 29

GMP 96

0.8 AMP + GMP

98 2 + 0.8

AMP + GMP 95 (94)

1 + 0.4 91

K326QP41OW AMP 2

site can restore low affinity binding of GMP to the mutant A site, another indication of synergism. The same pattern was observed for inhibition of the P410W C site mutant. Reduced inhibition resulting from defective binding of AMP to the mutant C site was largely restored by GMP since strong synergism was observed. This effect further supports the idea of synergism between nucleotide sites. The remaining data in Table I show that inhibition and synergism were not detected in the K326QP41OW double mutant.

Equilibrium Binding of Inhibitors-Equilibrium dialysis measurements were carried out to quantitate the binding of AMP and GMP to each of the amidotransferase nucleotide sites and to examine the possible interaction between the sites. Figs. 2 and 3 show the binding of AMP and GMP, respectively, to the wild type enzyme. The saturation curves given in Fig. 2 (top) and Fig. 3 (top) show binding of more than 1 eq of AMP and GMPhbunit. The number of AMP- and GMP-binding sites was determined from the Scatchard plots shown in the insets. Extrapolations give a binding stoichiometry of 1.9 AMP and 2.1 GMPhbunit . This stoichiometry reflects binding of each nucleotide to both the A and C sites. Binding constants calculated from the Scatchard plots are tabulated in Table 11. A single Kd of 611 p~ was calculated for interaction of AMP to the A and C sites in the wild type enzyme. The two sites to which GMP bound had different affinities (Fig. 3, top inset ), with Kd values of 31 and 122 p ~ . The higher affinity binding was assigned to the Asite and the lower affinity binding to the C site, based on the requirement of an intact A site for inhibition by GMP. Curvature in the Scatchard plot indicative of positive cooperativity is apparent at low GMP concentration for binding to the high affinity A site (Fig. 3, top inset). The 5-20-fold higher affinity for GMP relative to AMP binding accounts for the stronger inhibition by GMP compared with AMP. Since nucleotide binding was determined in the absence of PRPP, a substrate and competitor for inhibition, the simple quantitative relationship between nucleotide binding constant and inhibition constant is not meaningful.

To determine whether synergistic inhibition by AMP + GMP results from binding interactions, we examined the binding of one nucleotide in the presence of the other. The saturation data

f 1.40

1.20 ’

1 .oo

Y 0.004 I . . . . . . . . , , , , . , . , P

0 400 800 1200 1600 Free [AMP], UM

.5

.4

Y

~ ’ ~ - ~ . ’ . . ’ ” “ 4

0 200 400 600

Free [AMP], pM FIG. 2. Equilibrium binding of AMP to wild type enzyme. Top,

binding of AMP was determined in the absence of GMP. Bottom, binding of AMP in the presence of 350 PM GMP. The insets show Scatchard plots of the binding data. Y is fractional saturation, and AMP, is the concentration of free AMP.

in Fig. 2 (bottom) and the Scatchard plot in the inset show that GMP increased the affinity of AMP for one site by 10-fold and at the same time prevented binding of AMP to the second site. We consider it likely that GMP competes with AMP for binding to the A site and enhances binding of AMP to the C site. These results are summarized in Table 11. Similarly, AMP increased GMP binding affinity to one site by 4-fold and prevented GMP from binding to the other site (Fig. 3, bottom, and Table 11). I t is reasonable to infer that AMP bound to the C site and enhanced binding of GMP to the A site. There are five important conclusions from the binding of multiple nucleotides. (i) AMP and GMP compete for binding to both the A and C sites. (ii) GMP has a 4-fold higher affinity for the A site than the C site and a 20-fold higher affinity than AMP for the A site. (iii) AMP has comparable affinity for the two sites but in the presence of GMP, AMP binding to the C site is enhanced about 10-fold. (iv) Binding of AMP to the C site increased the affinity of GMP to the A site by 4-fold. (v) Enhanced binding of one nucleotide by the other explains synergistic inhibition by AMP plus GMP.


Free [GMP], pM

m

1 1.20 1

I m

)

0 200 400 600

Free [GMP], pM FIG. 3. Equilibrium binding of GMP to wild type enzyme. Top,

binding of GMP was determined in the absence ofAMP. Bottom, binding in the presence of 455 PM AMP. The insets show Scatchard plots of the binding data.

In a subsequent series of experiments, we determined binding of nucleotides to the enzymes having mutations in the A and C sites. The A site K326Q mutant had greatly reduced affinity for AMP as well as GMP. A binding curve for AMP is shown in Fig. 4. Binding of GMP was too weak to detect. Due to excessive scatter it was not possible to calculate binding stoichiometry from a Scatchard plot. The Kd value for AMP estimated from the saturation plot was -2.4 mM. The binding of AMP was arbi- trarily assigned to the non-mutant C site. The fact that the Asite mutation resulted in decreased binding not only to the defective A site but also to the intact C site further highlights the im- portance of interactions that lead to synergism. The binding of nucleotide mixtures provides direct confirmation for synergistic binding to the K326Q mutant. Low affinity binding of GMP to the mutant A site was obtained in the presence ofAMP (Fig. 5, top), and binding ofAMP to the C site was observed in the presence of GMP (Fig. 5, bottom). These results, summarized in Table 11, show that the K326Q mutation reduced the affinity for synergistic binding of GMP to the A site by more than 20-fold. Perhaps as a consequence, synergistic binding of AMP to the

TABLE I1 Equilibrium binding of nucleotides to wild type

and mutant amidotransferases Values for dissociation constant (K,) and nucleotide equivalents

bound at saturation ( n ) were obtained from Scatchard plots. All assign- ments of& and n to the A and C sites were based on the two-site model derived from analyses of nucleotide inhibition of wild type and mutant enzymes as well as labeling by nucleotide affinity analogs (Zhou et al., 1993).

Enzyme Nucleotide' K,,(nP

A site C site

Wild type AMP* w

611 (1.01 611 (1.0) AMP*/GMP (350 PM) NDr GMP*

64 (0.6) 31 (1.0) 122 (1.1)

GMP*/AMP (455 PM) 8 (0.9) NDc

K326Q AMP* ND AMP*/GMP (455 PM) ND

-2400

GMP* 219 (0.61

ND ND GMP*/AMP (455 PM) 184 (0.81 ND

P410W AMP* ND ND AMP*/GMP (455 MI ND 335 (0.71 GMP* 176 (1.4) ND GMP*/AMP (455 p ~ ) 11 (0.7) ND

The asterisk indicates the radioactive nucleotide. The concentra-

I, The extrapolated value n, equivalents bound, is given in parenthe- tion of unlabeled nucleotide competitor is given in parentheses.

ses. ND, binding not detected.

0 200 400 600

Free [AMP], pM FIG. 4. Binding of A" to the K326Q mutant enzyme.

intact C site was with 3-fold decreased afinity. A qualitatively similar pattern was observed for the P410W

C site mutant. The Kd for binding of individual nucleotides was increased relative to the wild type enzyme, but the P410W mutant still displayed synergism for binding nucleotide mixtures. Binding of AMP to the mutant C site was not detected, but low afinity AMP binding was restored in the presence of GMP (Fig. 6, top, Table 11). Binding of GMP to the A site was cooperative with a nearly 6-fold increase in Kd relative to the wild type, and this binding was enhanced to essentially the wild type affinity by AMP (Fig. 6, bottom, Table 11).

DISCUSSION In this study we have characterized the binding of purine

mononucleotides to the two nucleotide regulatory sites in E. coli glutamine PRPP amidotransferase. Direct evidence for two nucleotide regulatory sites in the E. coli enzyme was recently

6788 Glutamine PRPP Amidotransferase Nucleotide Regulatory Sites

" l . . . . . . . . . . . . . 0 200 400 600

Free [GMP], pM

'v .1 Y

o f . . . . . . . . . . . . . . 0 200 400 600

Free [AMP], pM

-1

FIG. 5. Binding of nucleotides to the K326Q mutant enzyme. Top, binding of GMP in the presence of 455 AMP. Bottom, binding of AMP in the presence of 455 GMP. Insets show Scatchard plots of the binding data.

obtained by affinity labeling experiments with nucleotide analogs and by mutagenesis (Zhou et al., 1993). The sites, which were unambiguously localized in the structural model of the homologous B. subtilis amidotransferase, are represented in Fig. 7. One site, defined genetically by mutations in several amino acid residues including LysaZ6, is required for inhibition of the E. coli enzyme by GMP. Amino acid side chains from two subunits contribute to binding of nucleotide to this site, designated the A (allosteric) site. Lys326 interacts with the a-PO; of nucleotide bound to the A site. The second nucleotide site, designated the C (catalytic) site, includes a conserved sequence (residues 365-375) and a nonconserved sequence (residues 408-413) (Makaroff et al., 1983). The conserved sequence has been implicated in PRPP binding in a family of phosphoribos- yltransferases (Hove-Jensen et al., 1986) and interacts strongly with ribose-5'-phosphate of the nucleotide in the C site of the B. subtilis enzyme (Fig. 7). The nonconserved sequence is near to, but does not directly contact, the base of the bound nucleotide in the B. subtilis structure. Nucleotide discrimination in the C site of the E. coli amidotransferase may be controlled by the

Y . . . . . . . . . . . . . .

0 200 400 600 Free [AMP], pM

1

0.00 200 400 600 . 800

Free [GMP], pM

0.70 1 > 8 I

> 0.30

0.20

0.10 Y

+ 0.00 100 200 300 400 500 600

Free [GMP], IJM FIG. 6. Binding of nucleotides to the P410W enzyme. Top, bind-

ing of AMP in the presence of 455 PM GMP. Center, binding of GMP in


408

Lys 326 ll

"A" site "C" site FIG. 7. Diagram of the A and C nucleotide-binding sites in glu-

tamine PRPP amidotransferase. Ca backbone traces are shown in purple for peptides in the A site and in black for peptides in the C site. Residues 364-379 are drawn beyond the consemed C site (365-375) to emphasize the P-loop-a structure of the PRPP-binding site. Molecules of GMP and AMP are bound to the A and C sites, respectively. Side chain atoms are shown for the mutated positions: K326 in the A site and P410 in the C site. Residue numbers are for the E. coli enzyme. The A site is between subunits in the tetrameric enzyme; primed residue numbers represent amino acids in a second subunit. The model is based on the crystal structure of B. subtilis amidotransferase, which has two molecules ofAMP bound. E. coli residues 258-262, 324-329, 364-379, and 407-413 correspond to B. subtilis residues 242-246,303-308,343-358, and 386-392, respectively. E. coli was modeled from B. subtilis S e P 9 .

nonconserved sequence. The P410W mutation in the nonconserved sequence was designed to obstruct binding of a purine base to the C site. We predicted that a rearrangement of the polypeptide backbone of the nonconserved sequence would be required to accommodate the substitution of Trp for and that this rearrangement would block the C site with the bulky Trp side chain. We therefore examined inhibition and nucleotide binding in the K326QA site mutant, P410W C site mutant, and the K326QP410W double mutant. Results of nucleotide inhibition and equilibrium binding to the wild type and mutant enzymes fully support the two-site model.

Enzyme inhibition and binding of individual nucleotides to the single site mutants demonstrate the selectivity of the A and C sites for GMP and AMP, respectively. The A site mutant was both insensitive to inhibition by GMP and bound GMP very weakly, thus establishing that binding of GMP to the A site is required for inhibition. Similarly, the C site mutant was par- tially insensitive to inhibition by AMP whereas inhibition by GMP was not affected. Thus, although AMP and GMP each bind to both sites in the wild type enzyme, perturbation of the A site is sufficient to specifically affect binding and inhibition by GMP and perturbation of the C site specifically affects binding and inhibition by AMP. Inhibition of the K326Q A site mutant by AMP but not by GMP presumably reflects the relative affinities of these nucleotides for the intact C site when the mutant A site is unoccupied. The considerably greater inhibition of the P410W C site mutant enzyme by GMP compared with AMP may reflect relative binding affinities of nucleotides

the absence of- and presence of 455 p~ AMP (bottom). Insets show Scatchard plots of the binding data.

to the intact A site when the mutant C site is unoccupied. On the other hand, weak inhibition by AMP may reflect residual binding of AMP to the mutant C site. The data in Table I1 show that the K d for GMP binding was 176 1.1~ whereas the Kd for AMP was too high to determine. It is not apparent how normal inhibition by GMP was maintained in light of the decreased binding affinity in this particular case.

Experiments with nucleotide mixtures provide strong evidence that synergistic inhibition results from the capacity of one nucleotide to enhance the binding of the other. Synergistic effects were seen in the wild type and in the mutants. In the wild type, GMP in the A site enhanced the affinity of AMP for the C site by nearly 10-fold. AMP bound to the C site enhanced the affinity of GMP for the A site by 4-fold. In the K326Q A site mutant, AMP increased the affhity of GMP for the mutant A site, and GMP increased the affinity of AMP for the intact C site. In the P410W C site mutant, GMP bound to the intact A site increased the affinity of AMP for the mutant C site, and AMP increased the affinity of GMP for the A site. In the K326Q/ P410W double mutant, neither binding nor inhibition were detectable for either nucleotide.

Our experiments show that the A and C sites are responsible for the synergistic inhibition of the E. coli enzyme by nucleotides. The proximity of the A and C sites in the B. subtilis structure explains that binding at one site might affect binding at the other, but does not provide a detailed structural explanation of the synergism in the E. coli enzyme. The proximity of the allosteric and catalytic sites is unlike other allosteric enzymes (Perutz, 1989) and undoubtedly is responsible for the highly unusual synergistic inhibition displayed by glutamine PRPP amidotransferase. Understanding the structural mechanism of allostery and of synergism in this amidotransferase will require knowledge of the structure of uninhibited enzyme and perhaps also of enzyme with inhibitor bound to only one site. Although we do not know the structural mechanism of the interactions detailed by this work, the physiological consequences of synergistic inhibition are apparent. In E. coli de nouo purine nucleotide synthesis is controlled by gene regulation and feedback regulation of glutamine PRPP amidotransferase. Gene regulation responds to levels of two purine bases, hypoxanthine and guanine (Rolfes and Zalkin, 1990; Houlberg and Jensen, 1983). Feedback regulation, on the other hand, senses the purine nucleotide end products. Maximal inhibition of the amidotransferase results when both nucleotides are in excess.

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Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzyrnol. 164,

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Makaroff, C. A,, Zalkin, H., Switzer, R. L., and Vollmer, S. J. (1983) J. Bid. Chern.

Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264,16613-16619 Messenger, L. J., and Zalkin, H. (1979) J. Bid. Chem. 264,33824392 Meyer, E., and Switzer, R. L. (1979) J. Biol Chem. 264,53974402 Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-236 Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642 Sanger, F., Nieklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,

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Zalkin, H., and Dixon, J. E. (1992) Prog. Nucleic Acids Res. Mol. Bid. 42,259-287 Zhou, G., Charbonneau, H., Colman, R. F., and Zalkin, H. (1993) J. Biol. Chern.

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Documents

Binding of Purine Nucleotides to Two Regulatory Sites Results in