Activation Mechanism and Modification Kinetics of Chinese Hamster Dihydrofolate Reductase by P-chloromercuribenzoate

Overview

Journal Biochem J

Specialty Biochemistry

Date 1998 Sep 22

PMID 9742228

Citations 1

Authors

J W Wu

Z X Wang

Affiliations

Soon will be listed here.

Abstract

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme-substrate complex, but is used to stabilize the enzyme-transition-state complex.

Citing Articles

Kinetic and stereochemical studies on novel inactivators of C-terminal amidation.

Feng J, Shi J, Sirimanne S, May S Biochem J. 2000; 350 Pt 2:521-30.

PMID: 10947967 PMC: 1221280.

References

Pace C . Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986; 131:266-80. DOI: 10.1016/0076-6879(86)31045-0. View

Duffy T, Sato J, Vitols K, Huennekens F . L1210 dihydrofolate reductase: activation and enhancement of methotrexate sensitivity. Adv Enzyme Regul. 1985; 24:13-25. DOI: 10.1016/0065-2571(85)90067-6. View

Stammers D, Champness J, Beddell C, Dann J, Eliopoulos E, Geddes A . The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme. FEBS Lett. 1987; 218(1):178-84. DOI: 10.1016/0014-5793(87)81042-6. View

Melera P, Davide J, Oen H . Antifolate-resistant Chinese hamster cells. Molecular basis for the biochemical and structural heterogeneity among dihydrofolate reductases produced by drug-sensitive and drug-resistant cell lines. J Biol Chem. 1988; 263(4):1978-90. View

Wang Z, Tsou C . Kinetics of substrate reaction during irreversible modification of enzyme activity for enzymes involving two substrates. J Theor Biol. 1987; 127(3):253-70. DOI: 10.1016/s0022-5193(87)80106-6. View

Tsou C . Kinetics of substrate reaction during irreversible modification of enzyme activity. Adv Enzymol Relat Areas Mol Biol. 1988; 61:381-436. DOI: 10.1002/9780470123072.ch7. View

Oefner C, Darcy A, Winkler F . Crystal structure of human dihydrofolate reductase complexed with folate. Eur J Biochem. 1988; 174(2):377-85. DOI: 10.1111/j.1432-1033.1988.tb14108.x. View

Prendergast N, Delcamp T, Smith P, Freisheim J . Expression and site-directed mutagenesis of human dihydrofolate reductase. Biochemistry. 1988; 27(10):3664-71. DOI: 10.1021/bi00410a022. View

Wang Z, Preiss B, Tsou C . Kinetics of inactivation of creatine kinase during modification of its thiol groups. Biochemistry. 1988; 27(14):5095-100. DOI: 10.1021/bi00414a022. View

10.

Wang Z . Two theoretical problems concerning the irreversible modification kinetics of enzyme activity. J Theor Biol. 1990; 142(4):551-63. DOI: 10.1016/s0022-5193(05)80108-0. View

11.

Davies 2nd J, Delcamp T, Prendergast N, Ashford V, Freisheim J, KRAUT J . Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate. Biochemistry. 1990; 29(40):9467-79. DOI: 10.1021/bi00492a021. View

12.

Cody V, Luft J, Ciszak E, Kalman T, Freisheim J . Crystal structure determination at 2.3 A of recombinant human dihydrofolate reductase ternary complex with NADPH and methotrexate-gamma-tetrazole. Anticancer Drug Des. 1992; 7(6):483-91. View

13.

Johnson B, Hecht M . Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology (N Y). 1994; 12(13):1357-60. DOI: 10.1038/nbt1294-1357. View

14.

Wang Z, Wang H, Zhou H . Kinetics of inactivation of aminoacylase by 2-chloromercuri-4-nitrophenol: a new type of complexing inhibitor. Biochemistry. 1995; 34(20):6863-8. DOI: 10.1021/bi00020a033. View

15.

Tsou C . Inactivation precedes overall molecular conformation changes during enzyme denaturation. Biochim Biophys Acta. 1995; 1253(2):151-62. DOI: 10.1016/0167-4838(95)00172-5. View

16.

Fan Y, Ju M, Zhou J, Tsou C . Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site. Biochem J. 1996; 315 ( Pt 1):97-102. PMC: 1217202. DOI: 10.1042/bj3150097. View

17.

HILLCOAT B, NIXON P, Blakley R . Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Anal Biochem. 1967; 21(2):178-89. DOI: 10.1016/0003-2697(67)90179-0. View

18.

Marangos P, Constantinides S . Factors affecting the folding and association of flounder muscle glyceraldehyde-3-phosphate dehydrogenase, in vitro. Biochemistry. 1974; 13(5):904-10. DOI: 10.1021/bi00702a012. View

19.

Koshland Jr D . Role of flexibility in the specificity, control and evolutiion of enzymes. FEBS Lett. 1976; 62(SUPPL):E47-52. DOI: 10.1016/0014-5793(76)80853-8. View

20.

KAUFMAN B, Kumar A, Blankenship D, Freisheim J . Activation of bovine and chicken liver dihydrofolate reductases and its relationship to a specific cysteine residue in their NH2-terminal amino acid sequences. J Biol Chem. 1980; 255(14):6542-5. View