Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.     

Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of "effective charge balance"

In thymidylate synthase, four conserved arginines provide two hydrogen bonds each to the oxygens of the phosphate group of the substrate, 2'-deoxyuridine-5'-monophosphate. Of these, R23, R178, and R179 are far removed from the site of methyl transfer and contribute to catalysis solely through binding and orientation of ligands. These arginines can be substituted by other residues, while still retaining more than 1% activity of the wild-type enzyme. We compared the kinetics and determined the crystal structures of dUMP complexes of three of the most active, uncharged single mutants of these arginines, R23I, R178T, and R179T, and of double mutants (R23I, R179T) and (R178T, R179T). The dramatically higher K(m) for R178T compared to the other two single mutants arises from the effects of R178 substitution on the orientation of dUMP; 10-15-fold increases in for R23I and R178T reflect the role of these residues in stabilizing the closed conformation of TS in ternary complexes. The free energy for productive dUMP binding, DeltaG(S), increases by at least 1 kcal/mol for each mutant, even when dUMP orientation and mobility in the crystal structure is the same as in wild-type enzyme. Thus, the four arginines do not contribute excess positive charge to the PO(4)(-2) binding site; rather, they ideally complement the charge and geometry of the phosphate moiety. More-than-additive increases in DeltaG(S) seen in the double mutants are consistent with quadratic increases in DeltaG(S) predicted for deviations from ideal electrostatic interactions and may also reflect cooperative binding of the arginines to the phosphate oxygens.     

Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.     

The functional role of tyrosine-200 in human testis angiotensin-converting enzyme.

The active site of angiotensin-converting enzyme (ACE) has been shown by chemical modification to contain a critical tyrosine residue, identified as Tyr-200 in human testis ACE (hTACE). We have expressed a mutant hTACE containing a Tyr-200 to Phe mutation. The mutant exhibits a marked decrease in kcat: 15-fold and 7-fold for the hydrolysis of furanacryloyl-Phe-Gly-Gly and angiotensin I, respectively, whereas its Km increases by only 1.6- and 2.2-fold, respectively. We conclude that Tyr-200 is not required for substrate binding. Instead, the effect on kcat together with a 100-fold decrease in affinity for the ACE inhibitor lisinopril indicates that Tyr-200 may participate in catalysis by stabilizing the transition state complex. Thus, Tyr-200 in hTACE has a role analogous to that of Tyr-198 in carboxypeptidase A.     

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.     

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.     

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.     

Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.     

Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.     

Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme.

The C-terminal residue of thymidylate synthase (TS) is highly conserved and has been implicated in cofactor binding, catalysis, and a conformational change. The codon for the C-terminal valine of Lactobacillus casei TS has been replaced with those for 19 other amino acids and the amber stop codon. Fourteen of the resulting mutant proteins were active by genetic complementation using a Thy- strain of Escherichia coli, and 18 mutants were active by in vitro assay. Only the aspartate and amber mutations had undetectable activity. All of the mutants were expressed at high levels (5-30% of soluble protein) and were purified by phosphocellulose chromatography. In general, the alterations at position 316 led to little effect on the Km for dUMP, an increase in Km for the folate cofactor, and a decrease in kcat. The observations show that TS can tolerate the substitution of most amino acids for valine at the C-terminus without a complete loss of activity, that hydrophobic substitutions are preferred, and that the C-terminal side chain is involved in both cofactor binding and catalysis. There was an excellent correlation between log kcat and hydrophobicity of the side chain at position 316 and an inverse correlation between log Km and the hydrophobicity of this residue. Kinetic parameters of the cofactor-independent TS-catalyzed dehalogenation of BrdUMP showed no variation with the side chain at position 316. In context of the structure of TS, it is proposed that binding of the cofactor triggers a conformational change in which the C-terminal side chain undergoes hydrophobic interactions that stabilize a rate-limiting transition state of the TS reaction.     

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.     

A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction

The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.     

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.     

Modification of a catalytically important residue of indoleglycerol-phosphate synthase from Escherichia coli

The active-site residues of indoleglycerol-phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol-phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide-directed mutagenesis. The specificity constant kcat/Km of the mutant is 3 x 10(4)-times lower than that of the wild-type enzyme, due to a 60-fold decrease in kcat and a 450-fold increase in Km. This finding shows that Lys 55 is important for both catalysis and substrate binding.     

Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access

Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.     

Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.