Kinetics of ligand binding to dihydrofolate reductase: binary complex formation with NADPH and coenzyme analogues

Dunn, Susan M. J.; Batchelor, John G.; King, Roy W.

doi:10.1021/bi00605a016

Cited by 79 publications

(97 citation statements)

References 19 publications

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“…A comparison of the data for MuDHFR, EcDHFR-i 9 36, and EcDHFR-i 7 136 (Figure 4) suggested that I 3 (I 2 for MuDHFR) was signi®-cantly populated at equilibrium. In fact, the rate constants obtained from the data described here suggested that for EcDHFR-i 9 36 the I 3 and N conformations were equally populated at equilibrium, and for MuDHFR, the I 2 conformation was populated at approximately 10% compared to N. It is known from previous work of the enzymatic properties of wild-type EcDHFR (Dunn et al, 1978;Penner & Frieden, 1987) that there exists two native forms of the enzyme which are signi®cantly populated at equilibrium. Since one form of the protein binds ligands tightly, one may measure the relative distribution of these two species and their rate of interconversion by monitoring the quenching of tryptophan¯uorescence upon the binding of NADPH (Cayley et al, 1981).…”

Section: A Native Form Of Dhfr May Be a Folding Intermediatementioning

confidence: 48%

GroEL-mediated folding of structurally homologous dihydrofolate reductases 1 1Edited by P. E. Wright

Clark

Frieden

1997

Journal of Molecular Biology

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Section: A Native Form Of Dhfr May Be a Folding Intermediatementioning

confidence: 48%

GroEL-mediated folding of structurally homologous dihydrofolate reductases 1 1Edited by P. E. Wright

Clark

Frieden

1997

Journal of Molecular Biology

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“…ThioNADP(H) has been used as an inactive cofactor for NADPH in experiments using chicken, mouse, Escherichia coli, and Lactobacillus casei, and it has been shown to bind to the cofactor binding site (Dunn et al, 1978;Smith and Burchall, 1983;Thillet et al, 1990;McTigue et al, 1993). However, when we tested the effect of NADPS on DHFR enzyme activity, only slight inhibition of human DHFR was observed even with 180 mM NADS or NADPS (data not shown).…”

Section: The Effect Of Nad/nadp Analogs On Dhfr Activity and Expressionmentioning

confidence: 85%

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Hsieh

Tedeschi²,

Lawal³

et al. 2012

Mol Pharmacol

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Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

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“…Steady-state kinetic parameters were obtained under the conditions of Stone and Morrison (12). Stopped-flow fluorescence quenching was carried out using the method of Cayley et al (13) (16), is greater than 300 s-1, in contrast to the 1.4 s-1 observed for wild type, and in parallel with the weak binding of H2F (Table 1 and see Table 3). Secondly, the deuterium isotope effect in V at pH values below the pKa for [Gly54]DHFR is 3.0, in contrast to a deuterium isotope effect in V of 1.1 for wild type.…”

mentioning

confidence: 99%

“…As shown in Table 2, the binding of NADPH, the rates of conformer interconversion, and their relative concentrations are all in good agreement with wild-type values, consistent with localized structural changes caused by the mutation. tRate of interconversion of E2 -* El (16), kobS for slow phase;…”

mentioning

confidence: 99%

Importance of a hydrophobic residue in binding and catalysis by dihydrofolate reductase.

Mayer

Chen

Taira

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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A conserved residue at the dihydrofolate binding site of dihydrofolate reductase (EC 1.5.1.3), leucine-54, was replaced with glycine to ascertain the role of this hydrophobic amino acid. The effect of the mutation is both to increase the dissociation rate of dihydrofolate and decrease the rate of hydride transfer thus changing the rate-limiting step in catalysis from product loss (leucine-54) to hydride transfer (glycine-54). The total stabilization by leucine-54 of the transition state for hydride transfer is ca. 104-fold (AAG 5.4 kcal/mol) at subsaturating dihydrofolate levels relative to free enzyme despite its location some 10 A from the site of chemical reaction.Classical mechanistic studies on enzyme catalysis have focused on active site residues involved as acid-base catalysts or in the formation of covalent intermediates. The importance of hydrophobic amino acids remote from or hydrophobic residues directly at the active site could not be readily ascertained. Site-directed mutagenesis provides a means to define the role of hydrophobic amino acids in enzymic catalysis. To date, the focus of most studies using site-directed mutagenesis remains on active site residues, deleting them or replacing amino acids with potentially more efficient catalysts (1-3). One striking mutagenesis study on t-RNATYr ligase (formerly t-RNATYr synthetase) (4) suggests the critical importance of remote amino acids in catalysis. Replacing two hydrogen-bonding residues in the binding site for the y-phosphate of ATP (nucleophilic attack occurs at the aPhosphate) decreased the rate of catalysis by a factor of 10 , while only weakening ATP binding by a factor of 5. We report here the effect on catalysis and binding of a mutation in dihydrofolate reductase (DHFR; EC 1.5. MATERIALS AND METHODSThe Gly-54 and Val-31 mutants were constructed by primer extension of oligonucleotides using as the template partially single-stranded pTY1 (8, 9), a derivative of pBR322 containing a 1-kilobase insert encoding forE. coli DHFR. The double mutant was constructed in the same manner, but using the Val-31 encoding plasmid as the template. The oligonucleotide sequence introduced a new restriction site (Fig. 1) 7718The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Kinetics of ligand binding to dihydrofolate reductase: binary complex formation with NADPH and coenzyme analogues

Cited by 79 publications

References 19 publications

GroEL-mediated folding of structurally homologous dihydrofolate reductases 1 1Edited by P. E. Wright

GroEL-mediated folding of structurally homologous dihydrofolate reductases 1 1Edited by P. E. Wright

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Importance of a hydrophobic residue in binding and catalysis by dihydrofolate reductase.

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