narrative from nih proposal (submitted in september, 1999)

RESEARCH PLAN Principal Investigator/Program Director (Last, First, Middle): Moe, Owen A.

Narrative from NIH Proposal (Submitted in September, 1999)

LEBANON VALLEY COLLEGE AS A SITE FOR AN R15 AREA GRANT

Background Information. Lebanon Valley College, an independent, comprehensiveliberal arts institution founded in 1866, enrolls over 1300 full-time students of which one thirdmajor in science or mathematics. Lebanon Valley College emphasizes quality teaching and takesseriously its mandate to educate students broadly in the liberal arts. The college has a regionalreputation for offering a rigorous academic program of high quality. U. S. News and WorldReport ranked Lebanon Valley among the top 10 regional liberal arts colleges in the north in its1998 - 2000 rankings.

The chemistry department at Lebanon Valley has been a pioneer in undergraduateresearch, sponsoring summer research programs for its majors every year for the past 50 years,with funding coming from both internal funds and external competitive grants. Of the studentsparticipating in the summer programs, greater than 75% have gone on to earn higher degrees ingraduate or professional programs. Recognition for the chemistry and biology programs atLebanon Valley College has come from:

▲ the Office of Scientific and Engineering Personnel of the National Research Council,which ranked the Lebanon Valley chemistry department among the top 4% and thebiology department among the top 10% of 877 undergraduate institutions nationwidein the number of graduates earning PhD degrees in the time period 1920-86.

▲ a 1987 study by the federal Office of Technology Assessment, which included LebanonValley College on its list of the nation’s 100 most productive institutions in scienceand engineering (Maxfield, B.D., Institutional Productivity: The UndergraduateOrigins of Science and Engineering PhDs, July, 1987, Office of TechnologyAssessment Contractor Report.)

The chemistry and biochemistry programs continue to earn external validation of theirresearch and educational initiatives. During the past eight years, chemistry and biochemistryfaculty members have been awarded 15 research and equipment grants totaling $393,000. In thesame time period, faculty in these programs won five educational grants totaling $398,000,supporting both curriculum development and faculty enhancement projects. In the past fouryears, faculty and student coauthors have produced twelve publications in research andeducational journals. Details on the grants and publications in chemistry and biochemistry areprovided on the departmental web site at http://www.lvc.edu/www/chemistry.

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Student Training in the Chemistry and Biochemistry Programs. The chemistryprogram at Lebanon Valley College has a staff of five full-time faculty members and is accreditedby the American Chemical Society. The biochemistry program is administered jointly withbiology, which has a staff of seven faculty. Since 1986, 44 chemistry and life science graduatesof Lebanon Valley College have earned PhD degrees, 21 in health-related sciences and 23 in thechemical sciences. In addition, 27 more recent graduates are currently pursuing PhD degrees. Since 1986, 42 Lebanon Valley graduates have earned doctoral degrees in the health professions.

Impact of Grant on Research Environment. The heart of the chemistry andbiochemistry programs at Lebanon Valley College is the annual summer research experience. Forthe past several years 8-10 students and 2-3 faculty have been engaged in summer research. While student research projects during academic semesters can achieve some results and givestudents a sense of research, there is no substitute for the full-time immersion in research thatthey experience during the summer months. Students have the time needed to perfect techniques,they learn to confront and solve problems, they begin to understand the power and limitations ofinstrumentation, and they learn how to evaluate, interpret, record, and present data. Theatmosphere is different during the summer - it is in one sense more relaxed, but there is also anadded importance to the work they are doing. Research students grow, becoming competent andconfident from the summer experiences. These students come back to Lebanon Valley College inthe fall and become leaders and motivators in our classrooms.

This AREA grant will strengthen the research environment at Lebanon Valley College bysupporting students involved in molecular biology, an entirely new area of research at LebanonValley College. While our biochemistry program does offer an upper-level molecular biologycourse, faculty at this institution have not yet engaged students in substantive research projectsin molecular biology. Dr. Patton’s two year appointment to the chemistry department offers aunique opportunity for current students to work with an experienced molecular biologist onactive site mapping by site-directed mutagenesis.

The fact that a research project, such as the one proposed here, has been peer reviewedand found worthy of support adds to its credibility in the eyes of the students, especially at asmall college. Students view their participation in such projects with pride and seriousness ofpurpose. The AREA grant will further support and motivate students by providing funds thatenable them to present their work at national venues. Students are excited by the tangibleoutcomes of research - publications and presentations. They will work long and hard to achieveresults that are worthy of public dissemination.

Institutional Support. For the past 25 years, Lebanon Valley College has been

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unwavering in its support of the sciences. The college’s commitment to scientific researchmanifests itself in two ways. First, the college provides room/board and stipends for studentsworking on summer research projects on campus, and has done so every summer for the past 25years. Secondly, the college strongly supports the acquisition of research grade instrumentation,both by providing matching funds for departmental grant proposals and by purchasingequipment outright. In 1999 for example, the college purchased a 300 MHz Bruker FT-NMR, aShimadzu spectrofluorometer, and a Hewlett Packard diode-array spectrophotometer, largelyfrom internal funds. The instrumentation that we have acquired recently, eleven majorinstruments in the past 14 years, has proved essential to our research programs in chemistry andbiochemistry. The college also provides for the purchase of some small equipment and suppliesfor research. For this AREA project, Lebanon Valley College will provide room/board forsummer research students during the summers of 2000-2002, and will provide additional studentstipends to allow a total of 5-7 students to work each summer with Drs. Moe and Patton.

RESEARCH PLAN

a. Specific Aims

Context. Guanosine-5´-monophosphate synthetase (EC 6.3.4.1), formerly calledxanthosine-5´-phosphate aminase, catalyzes the glutamine-dependent synthesis of 5´-guanosinemonophosphate (GMP) from 5´-xanthosine monophosphate (XMP) and 5´-adenosine triphosphate(ATP) (1):

XMP + ATP + glutamine + H

2 O Mg2 +

< GMP + AMP + PPi + glutamate

Several considerations make GMP synthetase (GMPS) from E. coli an important and instructivesystem for study. First, GMPS is a key biosynthetic enzyme, catalyzing the final step in the denovo pathway for the production of guanine nucleotides. Thus, GMPS activity is required toprovide precursors for the synthesis of RNA and DNA and to supply GTP for other essentialcellular processes such as protein biosynthesis, microtubule assembly, and activation of Gproteins. Secondly, it’s role in purine nucleotide biosynthesis makes GMPS an important targetenzyme for anticancer and immunosuppressive chemotherapeutics. Thirdly, the crystal structure ofGMPS reveals the presence of two independently-folded domains, a glutamine amidotransferasedomain and an ATP pyrophosphatase domain (2). Analyses of conserved sequence fingerprintsand of overall structure indicate that each domain is a member a broader enzyme family (2). Thus,experimental determinations related to protein structure, catalysis, or ligand binding for GMPSacquire an importance that transcends this single enzyme.

Project Goals. The research proposed here seeks to elucidate amino acid residuesinvolved at the binding sites for nucleotide substrates/products on the ATP-pyrophosphatasedomain of E. coli GMPS. The XMP/GMP site of GMPS cannot be defined from the x-raystructure because it is part of a region that is disordered in the crystal (2). The more clearly definedATP site involves a putative purine ring binding pocket and a conserved “P-loop” that interacts

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with the terminal pyrophosphate moiety of ATP (2). We will use the dual approach of site-directedmutagenesis and affinity labeling to identify and assess the function of specific amino acid residuesat the ATP and XMP/GMP binding sites of GMPS. Elucidation of specific amino acids will helpto define and characterize the XMP/GMP site, will extend conclusions previously drawn about theATP site, and will define catalytic and ligand binding roles of active site amino acids.

Affinity Labeling. We will build on initial affinity labeling experiments from ourlaboratory that have demonstrated inactivation of GMPS by reactive analogues of guanosine andadenosine nucleotides. Analogues tested thus far contain reactive labeling groups only in the 5´-phosphate portion of the nucleotide. We will use MALDI-TOF mass spectrometry to determine thestoichiometry of analogue incorporation in the modified enzyme. We will identify modified aminoacids through a process that employs enzymatic proteolysis, MALDI-TOF mass spectrometry ofthe resulting peptide mixture and, if needed, purification of radioactively-labeled peptides byHPLC with subsequent Edman sequencing. Furthermore, we will test another group of nucleotideanalogues, azido derivatives of ATP and GMP, which can function as photoaffinity labels. Because the reactive azido groups reside on the purine rings, the photoaffinity labels will probedifferent regions of the nucleotide binding sites.

Site-Directed Mutagenesis. We will complement and extend our previous work toinclude the production and analysis of site-directed mutants of GMPS. Through analysis of thecrystal structure of E. coli GMPS (2) and protein sequence alignments, we have identified 10highly-conserved amino acids that are likely to be located at or near the XMP/GMP site and 8residues at the ATP binding site. We will also construct site-directed mutants for residues that weidentify through affinity labeling. We will express and purify the mutants, analyze the functionaleffects of the mutations using enzyme kinetics and equilibrium ligand binding measurements, anduse that information to assess the roles of the mutated amino acids in nucleotide binding and/orcatalysis.

b. Background and Significance

Catalytic Mechanism. The reaction catalyzed by GMP synthetase involves replacementof an oxo group at the 2 position of the purine ring of XMP by an amino group as shown below:

OO3POH2C

OHOH

HHH H

N

N N

NH

O

O

XMP

2–OO3POH2C

OHOH

HHH H

N

N N

NH

O

NH 2

GMP

2–

H

The catalytic mechanism for this conversion involves an initial reaction between ATP and XMP toform pyrophosphate and an O2 -adenyl-XMP intermediate (3), as shown below. Ammoniaproduced from the hydrolysis of glutamine on the glutamine amidotransferase domain, orexogenous ammonia which can serve as a substrate in place of glutamine (1), then attacks the

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intermediate at the 2 carbon of the purine ring to yield GMP and AMP.

2–OO3POH2C

OHOH

HHH H

N

N N

NH

O

O OOH2C

OHOH

HHH H

P

O

O–

N

N N

N

NH2Adenyl-XMP Intermediate

Kinetic and isotope-exchange experiments involving E. coli GMPS indicate an orderedmechanism in which MgATP binds first, followed by XMP and then glutamine (4). A differentline of experimentation shows that XMP, in the absence of added MgATP or glutamine, binds toGMPS with a dissociation constant of ca. 100 µM and causes a conformational change thatprotects GMPS against inactivation by heat or proteolysis (5). However, the observation that theaddition of ATP or AMP/PPi lowers markedly the concentration of XMP needed to protect againstinactivation (5) is consistent with the ordered mechanism.

GMPS as a Chemotherapeutic Target. Much of the early interest in E. coli GMPSwas related to it’s role in the action of the antibiotic, psicofuranine. Psicofuranine, an analogueof the nucleoside adenosine, is a powerful and extremely specific inhibitor of bacterial GMPS (6). From its structure alone (structure given below), psicofuranine was expected to be a competitiveinhibitor versus ATP. A series of studies, however, demonstrated conclusively that psicofuraninebinds at an enzyme site that is separate from the XMP or ATP sites (7,8). Furthermore, XMP andpsicofuranine mutually enhance each other’s interactions with GMPS (7,9). The metabolic orregulatory significance, if any, of the psicofuranine site is unknown.

OHOH2C

OHOH

CH2OHHH H

N

N N

N

NH2Psicofuranine

Later work showed that GMP synthetase, as well as other enzymes in the de novo pathwayfor purine biosynthesis, are markedly elevated in rapidly proliferating cells such as those found inneoplastic tissues (10). Several lines of evidence argue that GMP synthetase is a promising targetfor anticancer and immunosuppressive therapies (11). For example, substituted derivatives of 2-benzamido-4-(isothiocyanatomethyl)-thiazole, which are potent inhibitors of GMP synthetase,provide strong antiproliferative activity against L1210 leukemic cells (12). Acivicin, an inhibitor ofGMP and CTP synthetases, depresses the growth of rat hepatoma cells (13, 14) and mizoribine-5-

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phosphate, an inhibitor of GMP synthetase and IMP dehydrogenase, functions as animmunosuppressant by blocking both humoral and cellular immunity (15). GMPS is thus a logicaltarget in any disorder in which a treatment strategy would involve lowering purine availability.

Crystal Structure of E. coli GMP Synthetase. The recently determined crystalstructure of E. coli GMP synthetase provides to date the only source of detailed structuralinformation about GMPS (2). While the enzyme exists as a dimer in solution (16), GMPScrystallizes as a tetramer, which is a dimer of dimers (2). The crystal structure confirms earlierconclusions (17), that the enzyme exists in two distinct structural and catalytic domains: aglutamine amidotransferase (or GAT) domain, and an ATP pyrophosphatase (or synthetase)domain. The GAT domain transfers ammonia from the amide group of glutamine to the synthetasedomain, where it adds to the activated O2–adenyl-XMP intermediate that is produced on thatdomain by a reaction between XMP and MgATP (3,4). The crystal structure indicates the presenceof a catalytic triad of Cys86 , His181, and Glu183 at the active site of the GAT domain (2). In theATP pyrophosphatase domain, the crystal structure reveals some details of a specific hydrophobicbinding pocket for the adenine ring of ATP, as well as a signature nucleotide-binding motif, or P-loop, for the binding of the β–γ pyrophosphoryl moiety of ATP (3).

Adenine Nucleotide Alpha Hydrolase Family. Comparisons of conservedsequence motifs in the putative binding site for ATP indicate that the ATP-pyrophosphatase domainis part of a newly-recognized family of adenine nucleotide alpha hydrolases (18). This familyincludes a sub-family of “N-type” ATP pyrophosphatases which form an activated adenylintermediate that is attacked by a nitrogen-based nucleophile (2). GMPS, NAD synthetase (19),arginosuccinate synthetase, and arginine synthetase comprise the N-type sub-family (2). Alphahydrolase family members that are not N-type ATP pyrophosphatases include phosphoadenylylsulfate reductase and ATP sulfurylases (18).

Human GMP Synthetase. Several recent reports detail the purification, cloning andexpression of human GMP synthetase (11, 20-22). Sequence comparisons indicate a high degreeof homology (41%) between the human and E. coli enzymes (2). Affinity labeling of the humanenzyme by acivicin caused modification of the active site residue, cys104, a conserved residue thatis homologous with cys86 of the catalytic triad of the E. coli enzyme (22). Although the human(M = 76,725; 693 amino acids) and E. coli (M = 58,604; 525 amino acids) enzymes differ in size,the high degree of sequence homology indicates that structural and functional aspects of thebacterial enzyme are likely to apply to the human enzyme (2). This conclusion is reinforced by ourown alignment analysis of the E. coli and human protein sequences, which demonstrates that thedifference in size is due to several large insertions that occur outside of the highly-conservedregions that are important for substrate binding and catalysis.

Nucleotide Binding Sites. One important structural aspect of E. coli GMP synthetasethat is not revealed by the crystal structure is the nature of the binding site for XMP on thesynthetase domain, a site that would be expected to also bind the product GMP that results fromamination of XMP. GMP synthetase was crystallized in the presence of ATP and XMP, butneither nucleotide was seen in the crystal structure. Only AMP and PPi were identified, suggestingthat ATP and XMP had reacted to form PPi and the O2–adenyl-XMP intermediate, whichsubsequently hydrolyzed to form AMP and XMP (2). The authors note that a phosphate residueseen in the crystal structure at the C-terminal end of the molecule is probably the 5´-phosphate of

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XMP, but that the rest of the XMP molecule, as well as a specific 22 amino acid portion of theATP pyrophosphatase domain that is also missing from the crystal structure, are disordered andimpossible to model (2). The 22-residue disordered peptide may be directly linked to XMPbinding since XMP protects against trypsin proteolysis at cleavage sites within the 22-residuesegment (5). For this enzyme, in which the crystal structure is not sufficiently detailed to definethe nature of the XMP/GMP binding site, the techniques of affinity labeling and site-directedmutagenesis can provide valuable auxiliary information.

In contrast to the XMP site, the adenosine ring and β–γ pyrophosphoryl portions of theATP binding site are more specifically defined by the crystal structure, to the point of suggestingsome possible interactions between specific amino acids and the nucleotide itself. Affinity labelingand site-directed mutagenesis can provide independent verification of amino acid-nucleotideinteractions at the ATP binding site, as it exists in the solution structure of the enzyme, and canelucidate other amino acids involved in ATP binding.

c . Preliminary Studies

Enzyme Purification. During the summer of 1997, at the beginning of our work onthis project, we purified native GMP synthetase in very low yield from E. coli strain B-96, apurine auxotroph that we grew under conditions that derepress the synthesis of GMPS (23). In thesummer of 1998, the laboratory of Dr. V. Jo Davisson of Purdue University generously sent us aculture of E. coli DH5α transformed with pguaA-tac, an expression plasmid encoding E. coliGMPS (24). We now purify in one week >100 mg of pure recombinant GMPS from 6-8 grams ofcell paste using streptomycin sulfate precipitation, DEAE Sepharose chromatography, andSephacryl S-200 chromatography. We assay enzyme activity spectrophotometrically, followingthe conversion of XMP to GMP at 290 nm (24) and we determine protein by the method ofBradford (25). The purified enzyme shows a single protein band on Coomassie-stained SDS gels.We dialyze purified protein versus buffers for the inactivation studies and store the dialyzedenzyme solutions at ca. -80 °C over liquid nitrogen in storage dewar.

Synthesis of Affinity Labels. We have synthesized two potential nucleotide-basedaffinity labels for the XMP/GMP binding site of GMP synthetase: 5´-[p-(fluorosulphonyl)-benzoyl]guanosine (5´-FSBG) and guanosine 5´-O-[S-(4-bromo-2,3-dioxobutyl)] thiophosphate(GMPSBDB). We use standard protocols developed by Dr. Roberta Colman at the University ofDelaware for these syntheses (26,27). We purchase one ATP analogue, 5´-[p-(fluorosulphonyl)-benzoyl]adenosine (5´-FSBA), from Sigma Chemical Corporation. Both 5´-FSBG and 5´-FSBAreact with enzyme nucleophiles through the displacement of fluoride from the fluorosulphonylgroup as shown below for 5´-FSBG.

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5´-FSBG

OOH2C

OHOH

HHH H

C

ON

N N

NH

O

NH2S

O

F

O

site of attack

GMPSBDB can react with enzyme nucleophiles through displacement of bromide, it can formhemiketals or thiohemiketals with alcohols and thiols, and it can react with arginine residues usingboth keto groups to produce a cyclic adduct (28).

GMPSBDB

OOH2C

OHOH

HHH H

PS

O

O–

CH2C

O

CO

CH2Br

N

N N

NH

O

NH2sitesof

reaction

Inactivation by Nucleotide Analogues. We have tested 5´-FSBG, GMPSBDB, and5´-FSBA, as potential affinity labels for GMP synthetase. The experimental approach that we takeand the type of data that we collect is virtually identical for each of the three reactive analogues. Therefore, we will present here detailed experimental results for 5´-FSBG only. A summary tablewill later compare final results for all three reagents.

To measure rate constants for enzyme inactivation, we incubate enzyme and 5´-FSBG in0.050 M PIPES buffer, pH 7.8 at 35 °C for 0 to 48 minutes. We remove aliquots at predeterminedtimes and assay the aliquots for enzyme activity remaining. A control sample containing enzymewith no 5´-FSBG is also monitored versus time. We express enzyme activity remaining as theratio, E/Eo, where E is the activity of the sample containing 5´-FSBG and Eo is the activity of thecontrol.

Data for inactivation of GMPS in the presence of three different concentrations of 5´-FSBGis shown in Figure 1. In this figure, the dashed line reflects the activity of the control which losesno activity under the conditions of the incubation. The concentrations of 5´-FSBG in the threeexperiments are: 0.063 mM (◆); 0.63 mM (▼), and 2.19 mM (▲). In all cases, 5´-FSBG showsmonophasic pseudo first-order inactivation kinetics. The observed rate constant for eachconcentration of 5´-FSBG, ko b s , is calculated from the slope of each first-order plot.

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Figure 1. First-Order Inactivation of GMP Synthetase by 5´-FSBG

-1.00

-0.75

-0.50

-0.25

0.25

ln [E/Eo]

0 10 20 30 40 50time, min

0.00

We have determined values for ko b s over a range of 5´-FSBG concentrations and plottedthe concentration dependence of 5´-FSBG inactivation in Figure 2. Figure 2. Effect of [5´-FSBG] on ko b s for Inactivation of GMP Synthetase

0.000

0.010

0.020

0.030

0 1 2 3 4 5[FSBG], mM

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The non-linear dependence of ko b s on [5´-FSBG] in Figure 2 is indicative of a model, outlinedbelow, in which 5´-FSBG forms a complex with the enzyme prior to the inactivation reaction:

E + FSBG E (FSBG) E SBG + HF

Kd k max

In this model, E–SBG is the covalent sulfonylbenzoylguanosyl-derivative of GMPS that resultsfrom an enzyme nucleophile displacing the fluoro group in 5´-FSBG. The kinetic parameter,kmax, is the first-order rate constant that results when the enzyme is fully saturated with 5´-FSBG.The rate law for this model is shown in Equation 1.

+ Kd

k maxk obs =[FSBG]

[FSBG] Equation 1

Data from Figure 2 were fit to Equation 1 and best-fit values for the two kinetic parameters weredetermined to be: Kd = 525 µM and kmax = 0.032 min - 1 . The solid line through the datapoints in Figure 2 is a theoretical curve generated using the best-fit parameters in Equation 1.

The two other nucleotide analogues that we have tested also inactivate GMP synthetase andthey exhibit saturation behavior similar to that seen in Figure 2 for 5´-FSBG. Table 1 summarizeskinetic parameters that we determined for inactivation by 5´-FSBG, GMPSDBD, and 5´-FSBA, aswell as the conditions under which they were measured. For GMPSBDB, the inactivation reactionis so fast that we need to reduce the temperature to 0°C to obtain measurable rate constants. Dissociation constants determined for the three nucleotide analogues are in the range of 358-550µM. These dissociation constants are somewhat higher than the Michaelis constants determinedfor the corresponding nucleotide substrates: Km = 45 µM for XMP and Km = 220 µM for MgATP(4). The concentration ranges used for the nucleotide analogues in our inactivation studies aresimilar to the ranges used with these analogues in other enzyme systems (29-31). Table 1. Inactivation Parameters for 5´-FSBG, GMPSBDB, and 5´-FSBA

Analogue Kd, µM kobs, min-1 Buffer Temperature

5´-FSBG 525 0.032 0.05 M PIPES, pH 7.84.5% dimethylformamide

35 °C

GMPSBDB 550 0.24 0.02 M PIPES, pH 7.4 0 °C

5´-FSBA 358 0.027 0.05 M HEPES, pH 8.44.5% dimethylformamide

35 °C

The reactive analogues that we use hydrolyze spontaneously during the inactivationexperiments. The rate constants for release of fluoride from 5´-FSBG and the release of bromidefrom GMPSBDB have been measured under conditions similar to those used in our experiments.

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For 5´-FSBG at 30°C in a barbital buffer at pH 7.65, the rate constant for the release of fluoride is0.0031 min-1 (32), which yields a reagent half-life of 223 min. Assuming a similar rate constantfor our system, only 14% of the 5´-FSBG reagent would be expected to hydrolyze during the 48minute time frame we used for the inactivation studies with 5´-FSBG. The stability of 5´-FSBAwould be expected to be similar to that of 5´-FSBG since they have the same reactivefluorosulfonyl group. The half-life of GMPSBDB in pH 7.0 PIPES buffer is 120 min (27),appreciably longer than the 0 to 40 min range that we used.

Effect of Active Site Ligands on Inactivation by Nucleotide Analogues. If anucleotide analogue forms a specific active-site complex with the enzyme prior to the inactivationreaction, we would expect that adding either the appropriate substrate or product to the inactivationmixture would slow the rate of inactivation through competition for the binding site. We measuredthe effect of active site ligands such as GMP, XMP, and ATP on the rate constants for inactivationof GMP synthetase by the three nucleotide analogues. In no cases did we observe protectionagainst inactivation by the active site ligands, alone or in combination.

Conclusions From Affinity Labeling Experiments. All of the nucleotideanalogues that we have tested inactivate GMPS, and all exhibit a concentration dependence that ischaracteristic of a site-specific binding step that precedes the inactivation reaction. These resultsare exactly what we would expect for an analogue binding at a nucleotide site. However, theinability of XMP, GMP, and ATP to protect against inactivation argues that the analogues may bebinding to some other site on the enzyme and inactivating by reacting at that site. It is notuncommon for affinity labels to react at other locations on an enzyme. The PI for this proposalfound, for example, that GMPSBDB reacted very rapidly with a surface cysteine ofadenylosuccinate synthetase, causing 50% inactivation. Only after that reaction had reachedcompletion was a much slower inactivation observed due to active site labeling (29).

Our operating hypothesis assumes that the nucleotide analogues that we have tested bind totwo types of sites on GMPS and react to covalently label an amino acid residue at each site. Kinetically, only inactivation at the first, rapidly-reacting site would be observable. A likelycandidate for the rapidly-reacting site would be the binding site for psicofuranine, an inhibitoryadenosine-like nucleoside described in the Background and Significance portion of thisproposal. Since the binding of psicofuranine itself causes a large increase in the affinity of theXMP site for XMP (7), it is reasonable to suggest that labeling the psicofuranine site with aguanosine-based affinity label might enhance a secondary labeling of the XMP/GMP binding siteby the same affinity label. Analyses by MALDI-TOF mass spectrometry and peptide sequencing,the costs for which will be supported by this grant, will allow us to test and develop thishypothesis, as described in the next section.

Structural Analysis of GMP Synthetase Sequences. The first step inconstructing site-directed mutants of E. coli GMP synthetase is to identify target amino acids ateach of the two nucleotide binding sites. To that end, we have analyzed an alignment of nineprokaryotic and lower eucaryotic GMP synthetases. ClustalW, a web resource of the HumanGenome Sequencing Center at Baylor College of Medicine (http://dot.imgen.bcm.tmc.edu:9331/multialign/multialign.html), was used in conjunction with BoxShade,from the ISREC Bioinformatics group in Switzerland (http://www.isrec.isbsib.ch:8080/software/BOX_form.html), to generate the sequence alignment. These sequences are retrievable from theSwissProt database using the following accession numbers: E. coli (P04079); H. influenzae

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(P44335); S. cerevisiae (P38625); H. pylori (O25165); A. aeolicus (O66601); B. subtilis(P29727); Synecho. sp. (P49057); C. ammoniagenes (O52831); and M. tuberculosis (Q50729). A high degree of sequence homology exists both in the glutamine amidotransferase domain and inthe ATP pyrophosphatase domain (hatched running line).

d. Research Design and Methods

This proposal, which requests funding for two years beginning in July, 2000, will supportresearch programs at Lebanon Valley College during the summer of 2001, and portions of thesummers of 2000 and 2002. Additionally, faculty and students involved will carry some of thesummer work into the intervening academic years. The approach described uses thecomplementary techniques of affinity labeling and site-directed mutagenesis. A summary outlineof the work planned and people involved is given in Table 2.

Table 2. Timetable for Proposed Research

Time Affinity Labeling Site-Directed Mutagenesis

Period Protein Constructs Assays/Analysis

Summer2000

Measure incorporation of FSBGand FSBA by MALDI-TOFPhotoaffinity labeling with

azido-ATP (Moe and 2 students)

Poly-HIS tagInitial mutants for

XMP siteExpression and

purification of mutants(Patton and 2 students)

Binding of AMP, PPiand ATP

(Moe, Patton, and 1-2student)

AcademicYear,

2000-2001

Synthesis of azido-GMPSynthesis of radioactive FSBG

and/or azido-ATP(Moe and 2 students)

XMP/ATP mutantsExpression and

purification of mutants(Patton and 2 students)

Binding of XMP(1 student)

Summer2001

Photoaffinity labeling Photoaffinity label incorporation

by MALDI-TOFHPLC with radioactive peptides

Sequencing of peptides(Moe and 2 students)

Mutants for ATP siteExpression and

purification of mutants(Patton and 2-3

students)

Analysis of purifiedmutants: kinetics and

binding studies(Patton, Moe, and 2

students)

AcademicYear

2001-2002

Continuation of summerprojects, as needed

(Moe and 1-2 students)

Protein construct workcompleted

Analysis of purifiedmutants

(Moe and 1 student)

Summer2002

HPLC with radioactive peptidesSequencing of peptides(Moe and 2 students)

Analysis of purifiedmutants

(Moe and 2 students)

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Affinity Labeling

Incorporation Experiments Using MALDI-TOF Mass Spectrometry. We willbegin by testing the hypothesis that 5´-FSBG binds and reacts at two distinct sites on each GMPSsubunit: a fast-reacting site that is not the XMP/GMP site, and a slower-reacting site which is theXMP/GMP site. It is possible that the fast-reacting site, the modification of which causes thekinetically-observable first-order inactivation of the enzyme, is the binding site for the antibiotic,psicofuranine. MALDI-TOF mass spectrometry has become a powerful technique for the analysisof chemically-modified proteins. If the molecular weight and amino acid sequence for a modifiedprotein are known, MALDI can determine the moles of reagent incorporated and can use molecularmass determinations to identify labeled peptides from a proteolytic digest of the modified protein.An excellent example of the use of MALDI-TOF mass spectrometry for active-site mappinginvolves affinity-labeled cytochrome bo3 from E. coli (33).

We will prepare 5´-FSBG-modified enzyme by reacting GMPS with 5´-FSBG until >98%inactivation has been achieved. The methods and conditions for inactivation will be the same asthose described in the previous section. We will dialyze the modified protein to remove excesslabeling reagent and submit a sample to the Macromolecular Core Facility of the Hershey MedicalCenter for analysis by MALDI-TOF mass spectrometry. A separate control enzyme will also beprepared using the same process, except that 5´-FSBG will not be present in the inactivationmixture. The Perceptive BioSystems Voyager MALDI-TOF mass spectrometer at HersheyMedical Center can measure protein molecular weight values to within 0.1%. An incorporation of1-2 mol reagent per mol GMPS monomer would produce an increase in mass of 0.8 - 1.5 %, wellabove 0.1% accuracy limit of the instrument.

If we find an incorporation approaching two moles of 5´-FSBG per mol of GMPS subunit,we will then run the modification reaction again, but in two different ways. First, we will addGMP or XMP to the inactivation mixture originally used, seeking to block incorporation of 5´-FSBG into the slowly-reacting active site by adding a natural ligand for that site. We may need toadd Mg2+ ion, pyrophosphate, and AMP along with the XMP if the reaction proceeds by thereported (4) ordered addition to the ATP site first, followed by XMP. If the second site is theactive site, we would expect to see by MALDI-TOF only one mole of 5´-FSBG incorporated permol subunit. In a second type of experiment, we would add psicofuranine to the originalinactivation mixture to block the rapidly reacting site. If the kinetically-observable inactivation isdue to modification of the psicofuranine site, the latter experiment should also show anincorporation of one mole of 5´-FSBG per mol of GMPS monomer.

If we measure incorporation consistent with two types of sites as described above, we willcarry out proteolysis of the 5´-FSBG labeled forms of the enzyme. We will need to test severalproteolytic enzymes (e.g., trypsin, chymotrypsin, endopeptidase from S.aureus, strain V8) andsets of reaction conditions to achieve as complete a digestion as possible. From the knownspecificity of the protease and the known sequence of E. coli GMPS, we can calculate molecularmasses of the peptides predicted for the proteolysis. We will work with a particular protease andnative GMPS until we have achieved the proper conditions for digestion and MALDI-TOFanalysis. We will then carry out mass spectral analyses of the peptide mixtures resulting fromproteolysis of the three modified forms of the enzyme. Any peptide carrying the 5´-FSBG labelwill show the predicted increase in mass. By this process, we should be able to identify the

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peptides that carry the amino acids that react with 5´-FSBG.

We will also apply MALDI-TOF to GMPS that has been modified by GMPSBDB and 5´-FSBA. The same procedure and approach that is used for 5´-FSBG will be used for GMPSBDBand 5´-FSBA. MALDI-TOF will allow us to pinpoint peptide sequences within which the variousaffinity labels reacted, but it will not tell us which specific amino acids reacted. Depending on thesizes of the peptides involved and the known reactivities of the amino acids present, we may beable to narrow down the site of reaction to a small number of residues or even identify it directly. Unambiguous determination of the labeled amino acids, however, will often require sequencing ofradioactively labeled peptides, as discussed later.

Photoaffinity Labeling. In addition to the three nucleotide analogues that we havetested, all of which carry reactive groups on the 5´ position of the ribose ring, we will initiate workwith photoaffinity labels that carry reactive azido groups on the purine ring. The labels we plan totest are 8-azido-ATP and 8-azido-GMP (34). Azido analogues offer several advantages as proteinlabeling reagents. Under conditions where they are not activated by light, the azido analogues canbe tested kinetically for their ability to inhibit GMPS competitively versus the substrates for whichthey serve as analogues. Competitive inhibition strongly indicates that an analogue binds at thedesired active site. Secondly, the pulse of UV light that activates the azido group is applied onlyafter the ligand has been given a chance to form an active site complex, thereby specificallytargeting active site residues. Non-specific labeling of amino acids on the protein can be reducedby placing a nucleophile in the buffer: upon light activation, affinity label not bound to the proteinwill react with the added nucleophile and not with surface nucleophiles on the enzyme. Thirdly,the presence of the azido group on the purine ring will allow us to probe the purine portions of thenucleotide binding sites of GMPS.

We will purchase 8-azido-ATP from ICN Biochemicals and will synthesize 8-azido-GMPusing published methods (34). In the case of the ATP site, we will use kinetic analysis to measurethe Ki for 8-azido-ATP versus ATP to confirm that inhibition is competitive. Using concentrationsof 8-azido-ATP greater than it’s Ki, we will attempt to modify the ATP binding site of GMPS byphotoaffinity labeling. The incubation mixture will include a weakly-nucleophilic buffer such asPIPES or HEPES, Mg2+, GMPS, 8-azido-ATP, and possibly an added nucleophile such as 2-mercaptoethanol. We have purchased and tested a water-cooled mercury vapor lamp/reactionchamber to produce the UV light pulse used for activation of the azido group. We will vary theduration of the light pulse, buffer pH, and amount of added nucleophile to achieve maximallabeling of the ATP site. Enzyme inactivation that accompanies labeling will be followed using thestandard spectrophotometric assay at 290 nm (23). The same approach will be taken to label theXMP/GMP site with 8-azido-GMP.

After demonstrating inactivation by a photoaffinity label, we will dialyze the modifiedenzyme to remove excess azido reagent. After dialysis, we will again test enzyme activity to insurethat we have achieved covalent labeling rather than having measured inhibition by a reacted form ofthe azido nucleoside. Modified GMPS prepared in this way will be submitted to theMacromolecular Core Facility of the Hershey Medical Center to determine the extent and locationof label incorporation by MALDI-TOF mass spectrometry as described earlier.

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Sequencing of Labeled Peptides. If the experiments with MALDI-TOF clearlydemonstrate active site labeling by analogues such as 5´-FSBG or 8-azido-ATP, we will work toidentify the amino acid residues involved. To do this, we will need radioactively-labeled forms ofthe affinity labels, two of which are available commercially: 8-azidoATP [α-32P] from ICN, and5´-FSBA [2,8-3H] from Moravek. We would synthesize radioactive forms of 5´-FSBG, 8-azido-GMP, and GMPSBDB from [8–3H] guanosine or [8–3H] GMP (Moravek) using proceduresdescribed earlier for the unlabeled analogues. The PI of this proposal synthesized [8–3H]GMPSBDB at the University of Delaware in 1995 and has experience in dealing with thecomplexities that attend a radioactive synthesis. Lebanon Valley College is licensed by the NuclearRegulatory Commission (license number 37-07710-02) for the use of 3H, 32P, and 14C.

The approach that we will take to label GMP synthetase and to separate and sequence thelabeled peptides is similar to the one taken by the PI of this proposal when working withadenylosuccinate synthetase and [8–3H] GMPSBDB (29). Our first experiments will be designedto measure incorporation of the radioactive affinity label into the enzyme. We will determineincorporation by incubating the enzyme with radioactive reagent under conditions identical to thoseused in the inactivation studies. We will remove aliquots of the reaction mixture at given timesduring the reaction, quench the inactivation reaction by adding 20 mM dithiothreitol, and separatethe labeled enzyme from the excess radioactive affinity label using either dialysis or gel permeationchromatography on a Sephadex G-50 column (32). Complete removal of excess affinity label willbe ensured by a second chromatographic separation on Sephadex G-50. Eluate from the secondcolumn will be analyzed for protein by the method of Bradford (25), for radioactivity by liquidscintillation counting (Packard Tri-Carb 4530), and for GMP synthetase activity. Incorporation,expressed as mol reagent incorporated per mol of enzyme subunit, will be determined both in thepresence and in the absence of the appropriate active site ligand (e.g., XMP, ATP, GMP).

We will correlate the time course of incorporation of radioactive reagent with the timecourse of inactivation to verify that these two processes occur in parallel fashion. Samples ofradioactively labeled GMP synthetase will be digested enzymatically using trypsin (Sigma) or theprotease from S. aureus, strain V8 (ICN). Enzymatic digests will be microcentrifuged at 14,000RPM to remove insoluble protein and the resulting supernatants will be filtered through a 0.45 µmMillipore filter to prepare them for HPLC. The stationary phase of our HPLC system is a 0.46-cmx 25-cm Supelcosil™ LC-318 C18 column with a 5-µ particle diameter and 300 Å pore size(Supelco). Separations will be carried out at a elution rate of rate of 1 mL/min in 0.1% aqueoustrifluoroacetic acid (solvent A) from 0 to 10 minutes, and then by a linear gradient from solvent Ato 85% solvent B (0.075% trifluoroacetic acid in acetonitrile) between 10 and 340 min. Detectionwill be carried out using a variable wavelength detector set at 215 nm. The gradient will bemodified as needed to achieve optimal separation. Samples of 1.0 mL will be collected and analiquot of each will be analyzed by liquid scintillation counting to determine radioactivity.

HPLC peptide elution profiles for GMP synthetase modified in the presence and absence ofactive site ligands will be compared to identify the peptides that are labeled only in the absence ofthe protecting ligands. We will freeze such peptide samples (HPLC fractions) in liquid nitrogenand transport them in a liquid nitrogen dewar to the core facility of the Hershey Medical Center,where they will be analyzed by MALDI-TOF mass spectrometry to determine the number ofpeptides in each HPLC fraction and their masses, from which peptide sequence identificationshould be possible. For those fractions that have ≤ 3 major peptides, we will carry out amino acid

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sequence analyses to determine the identity of the modified amino acid. The PI of this proposal,working with a protein of known sequence, has successfully sequenced modified peptides, even incases when multiple peptides were present in an HPLC fraction (29).

Site-Directed Mutagenesis

Addition of a Histidine Tag to GMPS. The purification of new protein constructsis often time-consuming, especially when multiple chromatographic steps are involved. In caseswhere a newly-constructed mutant protein is catalytically inactive, a multi-step purification processis difficult to complete without the ability to follow the protein. In order to accelerate and simplifypurification of our proposed constructs, we will add a six-residue polyhistidine tag to nativerecombinant GMPS and use that tag to effect a one-step purification via affinity chromatography(35). Dr. Patton, who has extensive experience in the construction and expression of recombinantproteins using T7 expression systems, will direct the subcloning of native, recombinant E. coliGMPS into the pET-30c(+) vector from Novagen (Milwaukee, WI). The advantage of using thisvector is the flexibility to generate either an N- or C-terminal His-tagged construct containing athrombin cleavage site. We choose to incorporate the tag at the N-terminus of GMPS where itshould have minimal impact on the structure and function of the ATP-pyrophosphatase domain.

The complete GMPS coding sequence, including start and stop codons, is easily removedfrom its current expression vector (pJF119EH) using EcoRI and HindIII. Ligation intoEcoRI/HindIII digested pET-30c(+) produces a vector that codes for the expression of a GMPSfusion protein that is tagged at the amino terminal end with both 6-His and S-tag sequences. Theidentification of positive clones will be confirmed using restriction digestion; protein will beexpressed using induction by isopropylthio-ß-D-galactoside (IPTG). With pET-30c(+) underkanamycin selection, we expect high-level protein expression in BL21(DE3) E.coli by avoiding β-lactamase degradation of ampicillin. Ni2+-affinity purified 6-His GMPS, judged pure byCoomassie-stained SDS gels, will be concentrated and dialyzed against buffer containing 15%glycerol prior to storage at -80°C. Assessment of the kinetic and nucleotide binding properties (seebelow) of wild-type 6-His GMPS will allow us to determine whether 6-His modification changesthe protein’s activity. If needed, the 6-His tag can be removed from the purified proteins withthrombin, or both tags can be removed with enterokinase. If we are unable to construct the fusionprotein without affecting it’s activity, the existing non-fusion construct will be used formutagenesis.

Construction of Site-directed Mutants. To construct site-directed mutants at theamino acid target residues that we identified earlier in the Preliminary Studies section, we willuse the Quick Change™ mutagenesis kit (Stratagene; La Jolla, CA), a technology that Dr. Pattonhas found to be reliable and rapid. In this thermal cycling method, a pair of complementary senseand anti-sense primers containing the desired base changes are extended and incorporated into newplasmids using the high-fidelity Pfu polymerase and a double stranded template. DpnI digestion ofthe resulting products removes the methylated parental plasmid leaving only the non-methylatedmutant constructs for bacterial transformation (36). Using this method, single amino acidreplacements occur with a routine efficiency of 95-100 %. Based on this high efficiency, and dueto the large number of constructs proposed, we will initially sequence only three transformants foreach site-mutant. Students routinely accomplish manual DNA sequencing with a high-degree ofsuccess using the dideoxy-based Thermosequenase™ kit (Amersham; Arlington Hgts, Ill). Dr.

PHS 398 (Rev, 4/98)


Patton has used this and similar approaches to generate site-directed constructs of guanine-nucleotide binding proteins (37-39).

In designing the mutants, we will determine substitutions for the targeted amino acids usingthe following criteria: (i) for target amino acids whose side chains contain polar groups potentiallyinvolved in ionic or hydrogen-bonding interactions, we will substitute with amino acids havingapproximately the same size/shape but lacking the same functionality (e.g., Phe for Tyr, Gln orMet for Lys or Arg, Val for Glu, Asp, or Thr, Ala for Cys or Ser, Leu for Asn or Gln); and (ii) fortarget amino acid with aliphatic or aromatic side chains potentially involved in ligand positioning,we will substitute with Ala.

Analysis of Expressed Mutants. Enzyme kinetics will serve as a primary tool foranalyzing site-directed mutants. We will measure maximal velocities and apparent Michaelisconstants for XMP and GMP by varying the two nucleotide substrates while holding glutamineconstant at an excess concentration, according to the method of von der Saal, et al,.(4). Formutations that produce changes in Michaelis constants for the nucleotide substrates, but cause littleor no diminution in maximal velocity, we will use the spectrophotometric assay at 290 nmdescribed earlier (23) for collecting kinetic data. The difference in molar absorptivity at 290 nmbetween XMP and GMP is 1500 M-1cm-1, making the assay only moderately sensitive. Formutants that decrease the maximal velocity, we will use a more sensitive coupled assay thatincludes the auxiliary enzymes AMP kinase, GMP kinase, pyruvate kinase, and lactatedehydrogenase (40). This coupled system generates 4 mol of NADH per mol XMP and producesa change in molar absorptivity of 2.49 x 104 M-1cm-1 at 340 nm, making it 16-fold more sensitivethan the assay at 290 nm.

We will also conduct ligand binding studies to assess the effect of mutations. We will useequilibrium dialysis to determine binding constants and the number of binding sites for theinteractions of ATP, XMP, AMP, and PPi with GMPS. The PI of this proposal has usedequilibrium dialysis in studies of thiamine diphosphate (14C) binding to the pyruvatedehydrogenase multienzyme complex (41) and in laboratory experiments (32P and 45Ca ligands)for our senior-level biochemistry laboratory course. We have constructed a set of microdialysiscells (100 µL) that can be assembled to run up to 20 determinations simultaneously, providingenough data points for two Scatchard plots. For the project proposed here, we will also testdisposable micro dialysis units (Slide-A-Lyzer® from Pierce) that will allow us to use as little as10µL of enzyme solution per determination. We will work with radioactive ligands to providehigh sensitivity in determining the concentration of free ligand at equilibrium. For binding studiesinvolving the XMP and ATP binding sites of GMPS we will purchase XMP[8-14C],ATP[2,8–3H], AMP[2,8-3H] (all from Moravek) and PPi[32P] (from NEN).

We will also explore the possibility of developing fluorimetric methods for ligand binding,as they may allow more rapid and sensitive determinations. Since XMP binding produces aconformational change large enough to protect GMPS against heat denaturation and proteolyticcleavage (5), it is likely that XMP binding produces a change in the intrinsic tryptophanfluorescence of the protein (5 Trp are located in the ATP pyrophosphatase domain). We willdetermine the enzyme-XMP dissociation constant by titrating GMPS with XMP and following thechange in fluorescence emission of Trp at 338 nm (42). Since XMP absorbs in the wavelengthregion used for excitation of tryptophan fluorescence, we will use triangular cells to measure

PHS 398 (Rev, 4/98)


fluorescence from the front inner surface of the cell, thereby avoiding inner filter effects caused byXMP absorbance. In the case of the ATP site, we will use fluorescence polarization (anisotropy)measurements (43) to determine the protein-ligand dissociation constant for ethenoadenosine-5´-triphosphate (εATP), a fluorescent ATP analogue. The dissociation constants for ATP, PPi, andAMP can then be measured using competition experiments.

Characterizing the protein constructs by using both ligand binding measurements andkinetic analysis will allow us to define with greater specificity the effects of the mutations onGMPS. For example, a particular mutant may be catalytically inactive, but still able to bind XMPand/or ATP. Ligand binding experiments provide the mechanism to test such a possibility, andprovide quantitative measures of the strength of binding at each site. Furthermore, by separatelymeasuring binding constants for PPi and AMP, we can assess the effects of mutations on twoseparate regions of the ATP binding site. Since Michaelis constants are complex composites ofrate constants for both binding and catalysis, they alone may not accurately reflect changes in thestrength of ligand binding.

To ensure that mutations have not perturbed the overall conformation of the enzyme, wewill submit samples of wild-type GMPS and all mutants to the Macromolecular Core Facility of theHershey Medical Center for analysis by circular dichroism (CD) spectroscopy.

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18. Savage, H., Montoya, G., Svensson, C., Schwenn, J. D., and Sinning, I. (1997). Crystalstructure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adeninenucleotide alpha hydrolases. Structure 5 , 895-906.

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21. Lou, L., Nakamura, J., Tsing, S., Nguyen, B., Chow, J., Straub, K., Chan, H., andBarnett, J. (1995). High-level production from a baculovirus expression system andbiochemical characterization of human GMP synthetase. Protein Expr. Purif. 6 , 487-95.

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24. Tesmer, J. J., Stemmler, T. L., Penner-Hahn, J. E., Davisson, V. J., and Smith, J. L.(1994). Preliminary X-ray analysis of Escherichia coli GMP synthetase: determination ofanomalous scattering factors for a cysteinyl mercury derivative. Proteins 18 , 394-403.

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33. Tsatsos, P. H., Reynolds, K., Nickels, E. F., He, D. Y., Yu, C. A., and Gennis, R. B.(1998). Using matrix-assisted laser desorption ionization mass spectrometry to map thequinol binding site of cytochrome bo3 from Escherichia coli. Biochemistry 37 , 9884-8.

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40. Spector, T. (1975). Studies with GMP synthetase from Ehrlich ascites cells. Purification,properties, and interactions with nucleotide analogs. J. Biol. Chem. 250 , 7372-6.

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narrative from nih proposal (submitted in september, 1999)

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