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X H Tan

Publications and source records attributed to X H Tan.

8 recordsLinked to original sources
PubMed1990

Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase.

Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of Km values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating Km and Kcat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The Km for NADPH for the K54Q mutant enzyme is 58-fold higher, while the Km for NADH for K54Q is only 3.9-fold higher than that of the wild type, indicating that the substitution of Lys-54 with Gln-54 decreases the apparent affinity of the enzyme for NADPH dramatically, but has a lesser effect on the apparent affinity for NADH.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Mapping the antigenic epitopes of human dihydrofolate reductase by systematic synthesis of peptides on solid supports.

All of the 181 possible overlapping hexapeptides as well as 179 octapeptides covering the amino acid sequence of human dihydrofolate reductase (hDHFR) were synthesized on polyethylene supports. The synthetic procedure of Geysen et al. (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274) was modified to obtain up to 100 nmol of peptide on each pin. Peptides constituting antigenic epitopes on hDHFR were identified by examining the binding of antibodies raised against both native and denatured hDHFR to these peptides by enzyme-linked immunosorbent assay. The peptides bound in a similar pattern to polyclonal antibodies against both native and denatured dihydrofolate reductase (DHFR). Six major epitopes were located corresponding to residues 27-33, 45-51, 67-74, 133-139, 153-158, and 176-181 using both hexapeptides and octapeptides. An additional epitope, constituting residues 14-21, was found by the use of octapeptides. Most of the epitopes are hydrophilic and reside largely in "loop" regions at the boundaries of secondary structural elements of hDHFR. This observation is consistent with our previous results which suggested that ligand binding at the active site of the enzyme can cause a dramatic reduction in antibody binding to DHFR due to conformational constraints in flexible loop regions in various parts of the molecule. The similarity of the immunogenic profiles of native versus denatured hDHFR indicates that the two forms of the antigen share the same amino acid sequence-specific epitopes. Competitive enzyme-linked immunosorbent assay showed that the binding of anti-hDHFR antiserum to both native and denatured hDHFR was inhibited by approximately 30% by the seven antigenic peptides, indicating that a significant proportion of the antibodies elicited by this enzyme is specific for short peptides. Besides revealing the antigenic structure of DHFR our results provide a rational basis for the design of mutant DHFRs to study the importance of loop residues in the conformational dynamics of the enzyme.

Amino Acid Sequence↗
PubMed1990

The importance of loop region residues 40-46 in human dihydrofolate reductase as revealed by site-directed mutagenesis.

Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible "loop" region between residues 40 and 46 of human dihydrolate reductase. Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) as well as the pH rate profile are virtually identical to that of the wild type. In contrast, the Vmax value of the mutant enzyme is decreased 2.5-fold. The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates. Agents such as KCl, urea, and organomercurials at concentrations which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant. Competitive enzyme-linked immunosorbent assay experiments using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking reduction in the binding of the antibodies. Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies. The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus "communicating" the effects of ligand binding.

Antibodies↗
PubMed1989

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase.

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Ligand-induced structural constraints in human dihydrofolate reductase revealed by peptide-specific antibodies.

Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats. Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111. The antisera were specific for the corresponding peptides used for immunization. Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays. However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody. Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR. Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site. Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1988

Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin.

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.

Aminopterin↗