Atomic structure of thymidylate synthase: target for rational drug design.

The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding.

Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177-->Asp).

BACKGROUND: Thymidylate synthase (TS) converts deoxyuridylate to thymidylate, an essential DNA precursor. Replacement of Asn177 with aspartate (Asn177-->Asp) in Escherichia coli TS creates a novel ability to methylate 2'-deoxycytidylate (dCMP). The dCMP-methylase activity of TS(Asn177-->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl donor. We have tested the possibility that the inactivation is due to negative cooperativity, created in the TS dimer by the Asn177-->Asp mutation. RESULTS: A heterodimeric form of TS, containing one wild type and one Asn177-->Asp active site, was created to test for negative cooperativity. Substrate inactivation still occurred, even with the mutation present at only one active site. CONCLUSIONS: Inactivation of TS(Asn177-->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation. The 'artificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other hetero-oligomeric proteins.

Systematic mutation of bacteriophage T4 lysozyme.

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Alteration of T4 lysozyme structure by second-site reversion of deleterious mutations.

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.

Structural aspects of the inhibition and catalytic mechanism of thymidylate synthase.

Thymidylate synthase (TS) is a target for anticancer drugs, due to its unique role in the biosynthesis of an essential DNA precursor. The X-ray structures available for several bacterial enzymes have been used to design novel inhibitors of TS, to structurally analyze the binding mode of existing inhibitors, and to propose catalytic roles for amino-acid residues on the protein. The first part of this paper describes some aspects of structure-based drug design, including a recent result from the groups of Montfort and Maley emphasizing the importance of conformational changes in inhibitor binding. The second part of the paper describes the work of the author on the TS mechanism, especially the catalytic roles of active site amino acids Asn177 and Glu58 in TS from Escherichia coli. An important function for Glu58 is proposed to be preventing the excessive stabilization of a covalent intermediate. The use of isotope effects to probe the mechanistic basis for stimulation of E. coli TS by magnesium ions, and to identify differences between the E. coli and human enzymes, is described. The hypothesis that N5 of tetrahydrofolate provides the basicity for deprotonation of the nucleotide is also discussed.

Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery.

Natural product analogs are significant sources for therapeutic agents. To capitalize efficiently on the effective features of naturally occurring substances, a natural product-based library production platform has been devised at Aurigene for drug lead discovery. This approach combines the attractive biological and physicochemical properties of natural product scaffolds, provided by eons of natural selection, with the chemical diversity available from parallel synthetic methods. Virtual property analysis, using computational methods described here, guides the selection of a set of natural product scaffolds that are both structurally diverse and likely to have favorable pharmacokinetic properties. The experimental characterization of several in vitro ADME properties of twenty of these scaffolds, and of a small set of designed congeners based upon one scaffold, is also described. These data confirm that most of the scaffolds and the designed library members have properties favorable to their utilization for creating libraries of lead-like molecules.

Kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4.

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate (CH2THF). The nucleotide analog 5-fluorodeoxuridylate (FdUMP) stoichiometrically inactivates CH by formation of a covalent complex containing enzyme, FdUMP, and CH2THF. Similar FdUMP complexes are formed by dTMP synthase and dUMP hydroxymethylase, enzymes which are homologous to CH. Both the association and the dissociation rate of the FdUMP complex are shown to be increased by the mutation of active site Asp179 to Asn. The mutated enzyme, CH(D179N), has an altered substrate preference, favoring dUMP rather than dCMP [Graves, K. L., et al. (1992) Biochemistry 31, 10315]. A value of 0.8 was determined for the alpha-secondary tritium equilibrium isotope effect on the binding of [6-3H]FdUMP to wild-type CH and to CH(D179N), using a mixture of 2-14C- and 6-3H-labeled FdUMP. These effects, similar to that found for TS, indicate that C6 of the nucleotide is saturated (i.e., sp3 hybridized) in the covalent complex of CH, FDUMP, and CH2THF. This strongly suggests that catalysis by CH proceeds via sequential sp2-->sp3-->sp2 hybridization changes at C6 of substrate nucleotides, and it is consistent with a transient covalent linkage of C6 to the thiol of an essential CH residue, Cys148. The values of the alpha-secondary 3H kinetic isotope effect (KIE) on kcat/KM for CH-catalyzed formation of Hm5dCMP caused by 6-3H-substitution of dCMP, with both wild-type CH and CH(D179N), were very close to 1.0. However, the KIE for CH-(D179N) with dUMP was 0.82.(ABSTRACT TRUNCATED AT 250 WORDS)

Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme.

Replacement of Asp20 in T4 lysozyme by Cys produces a variant with (1) nearly wild-type specific activity, (2) a newly acquired sensitivity to thiol-modifying reagents, and (3) a pH-activity profile that is very similar to that of the wild-type enzyme. These results indicate that the residue at position 20 has a significant nucleophilic function rather than merely an electrostatic role. The intermediate in catalysis by lysozyme is probably a covalent glycosyl-enzyme instead of the ion pair originally proposed.

Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis.

tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.

Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity.

The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn177, which was changed to aspartic acid. The side-chain amide of Asn177 forms hydrogen bonds with O4 and N3 of dUMP bound to the crystalline enzyme [Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, L., Maley, F. & Stroud, R. M. (1990) Biochemistry 29, 6964-6977]. This Asn is invariant in all natural sequences for TS known. The values of kcat for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kcat of wild-type TS with dUMP as substrate. TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-type TS turns over dCMP at < 10(-5) of its rate of dUMP turnover. Thus Asn177 is a major determinant of the pyrimidine nucleotide specificity of TS. The mutant enzyme, like wild-type TS, forms a covalent complex with 5-fluoro-dUMP in the presence of 5,10-methylenetetrahydrofolate. TS(N177D) also has a newly acquired ability to be transiently inactivated by dUMP. This time-dependent inactivation requires the presence of methylenetetrahydrofolate and may be due to the accumulation of the enzyme in the form of a catalytic intermediate. The likely mechanistic basis for discrimination by TS between dUMP and dCMP is their differing requirements for charge stabilization during covalent catalysis.

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I.

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.

pH dependence and solvent deuterium oxide kinetic isotope effects on Bacillus cereus beta-lactamase I catalyzed reactions.

The solvent kinetic isotope effects (SKIE's) on k(cat) (D(V)) and on k(cat/Km[D(V/K)] were determined for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of five substrates that have values of k(cat)/K(m) varying over the range (0.014-46.3) X 10(6)M(-1) s(-1) and of k(cat) between 0.5 and 2019 s(-1). The variation of D(V/K) was only from 1.06 to 1.25 among these compounds and that in D(V) was from 1.50 to 2.16. These results require that Dk(1), the SKIE on the enzyme-substrate association rate constant, and D(k-1/k2), that on the partition ratio of the ES complex, both be near 1. The larger SKIE observed on D(V) requires that an exchangeable proton be in flight for either or both the acylation and the deacylation reaction. The pH dependence of the values k(cat)/K(m) for three substrates shows identical pK(a)s of 5.5. and 8.4. This identity combined with the fact that only one of these three substrates is kinetically "sticky" proves that the substrates can combine productively with only one protonic form of the enzyme. There is considerable substrate variation in the pK(a) values of k(cat) observed vs. pH profiles; the inflection points for all substrates studied are at pH values more extreme than are observed in the pH profiles for k(cat)/K(m).

Disodium cromoglycate in treatment of asthmatic children.

In nearly all 38 children with asthma studied after administration of DSCG in Regina and Saskatoon, in our judgment there was marked improvement in chest symptoms, wheezing, coughing and weight gain. The patients and their families noted the same improvements. There was marked improvement in personality, school attendance, physical activity and other readily observed criteria. Other medications were sharply reduced in nearly every case. This was particularly significant in the case of steroid therapy and aerosol bronchodilators.DSCG has an important role in the treatment of properly selected cases of asthma. We noted no untoward side effects. It must be again emphasized that this drug is prophylactic and not palliative.

Isolation of a Staphylococcus aureus beta-lactamase-dicloxacillin complex and kinetic studies on the reactivation of the enzyme.

Exposure of the beta-lactamase from Staphylococcus aureus to the slowly reacting substrates cloxacillin or dicloxacillin results in time-dependent inactivation of the enzyme. Methods for the rapid separation of a beta-lactamase-dicloxacillin complex from excess inhibitor, using centrifuged columns of Sephadex G-25 or DEAE-Sephadex G-25, are described. The enzyme-dicloxacillin complex releases active enzyme, with specific activity identical to that of untreated enzyme, after storage at pH 7.5 at 15 degrees C. Full reactivation was accompanied by the release of 0.8 eq of hydrolyzed dicloxacillin. The complex is stable for up to 40 h when stored at pH 3 at 4 degrees C. The reactivation process, which occurs with first-order kinetics at 15 degrees C and pH values between 4 and 8, displays a pH dependence with apparent pKa's of 4.6 and 8.5, and a limiting value of the reactivation rate constant of 0.022 min-1. Deviation from first-order kinetics at pH 9 is consistent with a competing irreversible inactivation of the enzyme at that pH. This behavior differs substantially from that of the similarly inactivated beta-lactamase I from Bacillus cereus, whose rate of reactivation is independent of pH, but which undergoes irreversible denaturation at acidic pH [A. L. Fink, K. M. Behner, and A. K. Tan (1987) Biochemistry 26, 4248-4258]. Addition of hydroxylamine to the S. aureus beta-lactamase-dicloxacillin, complex stimulates the rate of reactivation by a maximum of 35%. This effect is hyperbolically dependent on the concentration of hydroxylamine with half-maximal stimulation at 2.8 mM. The Km for ampicillin hydrolysis catalyzed by the partially reactivated enzyme is identical to that measured for catalysis by the untreated enzyme. We discuss our observations in relation to models for the transient inhibition process.

Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase.

18O exchange experiments were designed to identify the final intermediate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase (CH). CH catalyzes the formation of 5-(hydroxymethyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF). CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. The final intermediate in the reaction catalyzed by TS or CH has been proposed to be the nucleotide with an exocyclic 5-methylene group covalently linked to the enzyme. This intermediate is then hydrated to HmdCMP (by CH) or reduced to deoxythymidylate (by TS). We report here that CH catalyzes the incorporation of 18O from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF). The cause of this exchange is a reverse reaction followed by a resynthesis. CH also catalyzes the exchange of 18O from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues that lack N-5. N-5 is the nitrogen that is likely to be bound to the methylene as it is transferred to dCMP. A CH variant that lacks the nucleophilic Cys 148 is incapable of promoting these 18O exchange reactions. The THF analogues lacking N-5 do not promote a CH-catalyzed reverse reaction. Rather, we propose that the CH-catalyzed 18O exchange reaction promoted by these THF analogues occurs via 5-methylene-dCMP linked to the enzyme through Cys 148. We conclude here that enzyme-bound 5-methylene-dCMP is the final intermediate during catalysis by CH, as has also been proposed for TS and dUMP.

Electrostatic guidance of catalysis by a conserved glutamic acid in Escherichia coli dTMP synthase and bacteriophage T4 dCMP hydroxymethylase.

Thymidylate synthase (TS) and dCMP hydroxymethylase (CH) are homologous enzymes which catalyze the alkylation of C5 of pyrimidine nucleotides. One of the first catalytic steps is isomerization of the alkyl donor, methylenetetrahydrofolate, from its N5,N10 bridged form to the N5 iminium ion upon enzyme binding. Glu58 in TS has been postulated [Matthews et al. (1990) J. Mol. Biol. 214, 937-948] to be involved in this isomerization and the deprotonation of C5 of the nucleotide. Substitution by Asp or Gln of Glu58 in Escherichia coli TS, or of the corresponding Glu60 in CH from phage T4, decreases the activity of either enzyme. Alkylation is slowed much more than deprotonation, indicating uncoupling of steps which are tightly coupled for the wild-type enzymes. The data support minor roles for Glu58/60 in nucleotide binding and in isomerization of methylenetetrahydrofolate, but no major roles in nucleotide deprotonation, product dissociation, or hydration catalyzed by CH. The primary role of Glu58/60 is to accelerate bond cleavage between N5 of tetrahydrofolate and the methylene being transferred. The influence of Glu58/60 on the rate of bond cleavage is proposed to arise from electrostatic destabilization due to the proximity of the glutamyl carboxylate, of the anionic species formed when C5 of the nucleotide is deprotonated. The proposal explains the uncoupling of deprotonation and alkylation with the Glu58/60 variants and the reduced kinetic isotope effect on hydride transfer for TS(Glu58Gln). The inability of 5-deazatetrahydrofolate to stimulate enzyme-catalyzed tritium exchange from [5-(3H)]nucleotides into solvent suggests that N5 of tetrahydrofolate is the base which deprotonates the nucleotide.

Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.

The proposed roles of Cys148 and Asp179 in deoxycytidylate (dCMP) hydroxymethylase (CH) have been tested using site-directed mutagenesis. CH catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate. CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. Conversion of Cys148 to Asp, Gly, or Ser decreases CH activity at least 10(5)-fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys residue in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of the substrate dUMP to the side-chain amide of an Asn; the corresponding residue in CH is Asp179. Conversion of Asp179 to Asn reduces the value of kcat/KM for dCMP by (1.5 x 10(4))-fold and increases the value of kcat/KM for dUMP by 60-fold; as a result, CH(D179N) has a slight preference for dUMP. Wild-type CH and CH(D179N) are covalently inactivated by 5-fluoro-dUMP, a mechanism-based inactivator of TS. Asp179 is proposed to stabilize covalent catalytic intermediates, by protonating N3 of the pyrimidine-CH adduct.

New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity.

Leishmania and other trypanosomatid protozoa require reduced pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for effective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts- knockouts) do not survive in animal models, validating this enzyme as a target for effective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and/or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1.

Anomalous pH dependence of the reactions of carbenicillin and sulbenicillin with Bacillus cereus beta-lactamase I. Influence of the alpha-substituent charge on the kinetic parameters.

The pH dependence of k(cat) for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of carbenicillin(VI), which differs from benzylpenicillin (I) in having a carboxylic moiety alpha to the phenyl ring, exhibits a profile consistent with a model in which the alpha-COOH and alpha-COO forms of the ES complex turn over with respective rate constants of 2152 s(-1) and 384 s(-1). The pK(a)(app) for the alpha-COOH is shifted from 3.2 in solution to 6.1 in the ES complex. The normalized k(cat)/K(m) vs. pH profile for VI is not superimposable on that of I, indicating that both the neutral and anionic forms of the carboxyl moiety of VI combine with the enzyme to give the first irreversibly formed complex, presumably the acyl-enzyme. Quantitative accord with the kinetic data is achieved only through fitting to a model where kinetically significant proton transfer in the ES complex is permitted. The second-order rate constants for the reaction of the enzyme with the alpha-COOH and alpha-COO forms of VI are 2.2 x 10(8) M(-1) s(-1) and 3.8 x 10(6) M(-1) s(-1), respectively. The high value for the alpha-COOH form suggests that this reaction may be in part diffusion controlled. This conjecture is borne out by the observation that the sensitivity of k(cat)/K(m) to eta(rel) decreases with increasing pH for VI, whereas this sensitivity is pH independent for I. These conclusions are further supported by the results of a kinetic investigation of the pH dependence of sulbenicillin (VII) where an alpha-SO3H replaces the alpha-COOH of VI. The strongly acidic sulfonic acid moiety of VII is fully ionized throughout nearly the entire pH range of interest, and its kinetics, as a function of pH, are very similar to those observed and calculated for the alpha-COO form of VI. Solvent deuterium kinetic isotope effects are reported for k(cat) and k(cat)/K(m) for both VI and VII.

The roles of pteridine reductase 1 and dihydrofolate reductase-thymidylate synthase in pteridine metabolism in the protozoan parasite Leishmania major.

Trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. A broad spectrum pteridine reductase (PTR1) was recently identified in Leishmania major, whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. The properties of PTR1 suggested a role in essential pteridine salvage as well as in antifolate resistance. To prove this, we have characterized further the properties and relative roles of PTR1 and dihydrofolate reductase-thymidylate synthase in Leishmania pteridine metabolism, using purified enzymes and knockout mutants. Recombinant L. major and Leishmania tarentolae, and native L. major PTR1s, were tetramers of 30-kDa subunits and showed similar catalytic properties with pterins and folates (pH dependence, substrate inhibition with H2pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies of ptr1(-) and dhfr-ts- mutants implicated only PTR1 in the ability of L. major to grow on a wide array of pterins. PTR1 exhibited 2000-fold less sensitivity to inhibition by methotrexate than dihydrofolate reductase-thymidylate synthase, suggesting several mechanisms by which PTR1 may compromise antifolate inhibition in wild-type Leishmania and lines bearing PTR1 amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen.