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H T Spencer

Publications and source records attributed to H T Spencer.

At least 19 recordsLinked to original sources
PubMed2000

Catalytic cysteine of thymidylate synthase is activated upon substrate binding.

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

Binding Sites↗
PubMed2000

Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.

Amino Acid Substitution↗
PubMed1999

Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.

Bacterial Proteins↗
PubMed1999

Ligand-mediated induction of thymidylate synthase occurs by enzyme stabilization. Implications for autoregulation of translation.

Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N5, N10-methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.

Base Sequence↗
PubMed1999

Substitution at residue 214 of human thymidylate synthase alters nucleotide binding and isomerization of ligand-protein complexes.

Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089-7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme-substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, Kiso, the ratio of the forward rate of isomerization (kiso) to the reverse rate of isomerization (kr, iso), is 2-6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on kiso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.

Amino Acid Substitution↗
PubMed1999

Mechanisms of acquired resistance to thymidylate synthase inhibitors: the role of enzyme stability.

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrimidines 5-fluorouracil and 5'-fluoro-2'-deoxyuridine (FdUrd) or the antifolates AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of cancer, particularly cancer of the colon and rectum. Numerous studies have shown that TS gene amplification, leading to mRNA and enzyme overproduction, is a major mechanism of resistance to these inhibitors. In the present work, we have isolated and characterized FdUrd-resistant derivatives of several human colon tumor cell lines. Although gene amplification was commonly observed, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to be caused by expression of a metabolically unstable TS molecule. The reduced half-life of TS in this line was caused by a Pro-to-Leu substitution at residue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resistant line, which had an excess of enzyme relative to mRNA, the TS molecule was more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that the stabilization must be caused by changes in one or more cellular factors that regulate TS degradation. The results indicate that changes in the stability of the TS polypeptide accompany, and even contribute to, acquired resistance to TS inhibitors in colon tumor cells.

Amino Acid Substitution↗
PubMed1998

A structural role for glutamine 214 in human thymidylate synthase.

Studies of the crystal structures of thymidylate synthase (TS) have revealed that a kink is present in beta-sheets that form the core of the enzyme. The beta-kink is proposed to serve as a "hinge" during conformational changes that occur in the enzyme after ligand binding at the active site. A residue in one of the beta-bulges that form the kink, glutamine at position 214 of human TS, is highly conserved in all TSs and is postulated to interact with nucleotide ligands that bind at the active site. To examine the role of this residue, glutamine at position 214 was replaced by residues that differ in volume, hydrophobicity, electrostatic charge, and hydrogen bonding potential. Genetic complementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chain volumes or that are prohibited in beta-bulges created loss of function proteins. Kinetic studies indicated that residue hydrophobicity is not correlated with catalytic activity. Residues that are predicted to alter the charge at position 214 created enzymes with kcat/Km values at least 10(3) lower than those of the wild type. Kinetic and ligand binding studies indicated that residue 214 is involved in nucleotide binding; however, hydrogen bonding potential does not contribute significantly to nucleotide binding energy. The data are consistent with the hypothesis that residue 214 is involved in maintaining the enzyme in a conformation that facilitates nucleotide binding and catalysis.

Amino Acid Substitution↗
PubMed1998

Retroviral coexpression of thymidylate synthase and dihydrofolate reductase confers fluoropyrimidine and antifolate resistance.

Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. We show that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents. Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold). Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30- to 140-fold). A L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA that encodes both TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents.

Animals↗
PubMed1997

Comparison of the protection of cells from antifolates by transduced human dihydrofolate reductase mutants.

Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.

Aminopterin↗
PubMed1997

Sensitization of hematopoietic stem and progenitor cells to trimetrexate using nucleoside transport inhibitors.

Antifolates such as methotrexate (MTX) and trimetrexate (TMTX) are widely used in the treatment of cancer and nonmalignant disorders. Transient, yet sometimes severe myelosuppression is an important limitation to the use of these drugs. It has previously been shown that clonogenic myeloid progenitors and colony-forming units-spleen are resistant to antifolates, suggesting that myelotoxicity occurs late in hematopoietic development. The goal of this study was to define the mechanisms by which primitive hematopoietic cells resist the toxic effects of antifolate drugs. To test the hypothesis that myeloid progenitors may salvage extracellular nucleotide precursors to resist TMTX toxicity, a defined liquid culture system was developed to measure TMTX toxicity in expanding progenitor populations. These in vitro experiments showed that both human and murine progenitors can resist TMTX toxicity by importing thymidine and hypoxanthine from the serum. As predicted from these findings, several drugs that block thymidine transport sensitized progenitors to TMTX in vitro, although to differing degrees. These nucleoside transport inhibitors were used to test whether progenitors and hematopoietic stem cells (HSCs) could be sensitized to TMTX in vivo. Treatment of mice with TMTX and nitrobenzylmercaptopurineriboside phosphate (NBMPR-P), a potent transport inhibitor, caused significant depletions of clonogenic progenitors within the bone marrow (20-fold) and spleen (6-fold). Furthermore, NBMPR-P administration dramatically sensitized HSCs to TMTX, with dual-treated mice showing a greater than 90% reduction in bone marrow repopulating activity. These studies demonstrate that both myeloid progenitor cells and HSCs resist TMTX by using nucleotide salvage mechanisms and that these pathways can be pharmacologically blocked in vivo using nucleoside transport inhibitors. These results have important implications regarding the use of transport inhibitors for cancer therapy and for using variants of dihydrofolate reductase for in vivo selection of genetically modified HSCs.

Animals↗
PubMed1997

A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs.

Chemoresistance gene transfer is an experimental method to protect hematopoietic cells from the toxicity of anticancer drugs. Because multiple drugs are usually given together in cancer therapy, this strategy will ultimately require vectors expressing multiple chemoresistance genes. For this reason, we designed a bicistronic retroviral vector (HaMID) containing a modified human multidrug resistance-1 cDNA and a mutant human dihydrofolate reductase cDNA bearing a leucine to tyrosine substitution at codon 22 (L22Y). To determine if this vector would confer dual drug resistance to hematopoietic cells, recombinant retrovirus was used to transduce the human CEM T lymphoblastic cell line as well as primary murine myeloid progenitors. Growth suppression assays, using polyclonal transduced CEM cells, demonstrated increased resistance to taxol (13-fold), trimetrexate (8.9-fold), vinblastine (5.6-fold), methotrexate (2.5-fold), and etoposide (1.5-fold) when used as single agents. HaMID-transduced cells also grew at a logarithmic rate in the simultaneous presence of 25 nM taxol and 100 nM trimetrexate while control cells were entirely growth inhibited by this drug combination. Similarly, HaMID-transduced murine myeloid progenitors acquired increased resistance to taxol (2.9-fold) and trimetrexate (140-fold), and were able to form colonies in the simultaneous presence of both drugs. Our results suggest that retroviral transfer of HaMID into primary hematopoietic cells should reduce the myelosuppression associated with the combined use of antifolates and P-glycoprotein-effluxed drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Kinetic scheme for thymidylate synthase from Escherichia coli: determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis.

We have determined kinetic and thermodynamic constants governing binding of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + dUMP --> TS x dUMP + (6R)-5,10-CH2-H4folate --> TS x dUMP x (6R)-5,10-CH2H4folate --> TS x dTMP x H2folate --> TS x dTMP --> TS as predicted previously by others from steady-state measurements. (2) When substrates are saturating, the overall reaction rate is governed by the slow conversion of enzyme-bound substrates to bound products as demonstrated by (i) large primary and secondary isotope effects on k(cat) and (ii) high rates of product dissociation compared to k(cat). (3) Stopped-flow studies measuring the binding of 10-propargyl-5,8-dideazafolate, an analog of (6R)-5,10-CH2H4folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physical events corresponding to spectral changes which are observed with the wild-type enzyme during initiation of catalysis. A kinetically identifiable reaction step, TS x dUMP x (6R)-5,10-CH2H4folate --> (TS x dUMP x (6R)-5,10-CH2H4folate)*, likely represents reorientation of the C-terminus of the enzyme over the catalytic site. This seals the substrates into a relatively nonaqueous environment in which catalysis can occur. (4) Although TS is a dimer of identical subunits, catalysis is probably confined to only one subunit at a time. (5) The "high-resolution" kinetic scheme described herein provides a framework for the interpretation of the kinetics of catalysis by mutant ecTS chosen to provide insights into the relationship between structure and function.

Catalysis↗
PubMed1996

A gene transfer strategy for making bone marrow cells resistant to trimetrexate.

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

Animals↗
PubMed1996

Mutations in the gene for human dihydrofolate reductase: an unlikely cause of clinical relapse in pediatric leukemia after therapy with methotrexate.

Resistance to methotrexate (MTX) in some sublines of mammalian cells is reported to be due to one of the following amino acid substitutions in dihydrofolate reductase (DHFR) that lower inhibition by MTX: Gly15 to Trp, Leu22 to Arg or Phe or Phe31 to Trp or Ser. We have produced variants of human DHFR (hDHFR) with these substitutions by directed mutagenesis. Recombinant hDHFR variants expressed in Escherichia coli have greatly decreased inhibition by MTX, but decreased catalytic efficiency, and in one case decreased stability. When a retroviral vector encoding wild-type (wt) hDHFR or one of these variants was introduced into murine fibroblasts or bone marrow progenitors, modest protection from MTX was conferred, even by wt. Relapsed pediatric patients with acute lymphoblastic leukemia who have received multiple courses of high-dose MTX seem most likely to develop such MTX resistance. cDNA was reverse transcribed from blast mRNA from 17 of these patients. However, upon amplification and sequencing of DHFR cDNA, no resistance mutation was found. The explanation for this probably lies in the need for considerable gene amplification to offset lowered catalytic efficiency, and the need for two-base changes for most substitutions, both of which are probably infrequent events.

Adolescent↗
PubMed1995

Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers.

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

Amino Acid Sequence↗
PubMed1994

Critical role of phenylalanine 34 of human dihydrofolate reductase in substrate and inhibitor binding and in catalysis.

Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The variant enzymes are stable and have decreased affinities for methotrexate (by factors of 2700-60000 at pH 7.65) due to a decreased rate of methotrexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether these enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marked inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry and fluorimetry and other methods, it has been shown that, like the wild-type enzyme, the variants have a branched reaction pathway, but in contrast to the wild-type enzyme, the distribution of flux between alternate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociation of tetrahydrofolate from the ternary product complexes of the variant enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the resulting abortive complex. When steady state kinetics for this model are simulated using the experimentally determined rate and dissociation constants for the alanine 34 variant, most steady state experimental results are closely approximated.

Ammonium Sulfate↗