PMID: 2505841Jul 11, 1989Paper

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

Biochemistry
J AndrewsS J Benkovic

Abstract

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid s...Continue Reading

References

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Citations

Jul 1, 1990·Biochemical Pharmacology·J Feeney
Feb 1, 1996·Molecular and Biochemical Parasitology·P RecheL M Ruiz-Perez
Jul 20, 1994·Biochimica Et Biophysica Acta·S P SassoC M Briand
Dec 9, 2010·Biochemistry·Clarissa M CzeksterJohn S Blanchard
Dec 17, 2011·Journal of the American Chemical Society·Vanja StojkovićAmnon Kohen
May 21, 2003·Proceedings of the National Academy of Sciences of the United States of America·Thomas H RodCharles L Brooks
Apr 10, 2015·Physical Chemistry Chemical Physics : PCCP·Louis Y P LukRudolf K Allemann
Jul 12, 2014·Biochemistry·Enas M BehiryRudolf K Allemann
Jan 13, 2006·Journal of Molecular Biology·Vladimir I PolshakovJames Feeney
Oct 7, 2004·Applied and Environmental Microbiology·Angela MatthewsVladimir Jiranek
Jan 2, 2010·The Journal of Physical Chemistry. B·Kei MoritsuguJeremy C Smith
Jul 23, 2008·Biochemistry·Thomas V RieraLizbeth Hedstrom
Mar 7, 1997·Journal of Molecular Biology·C S VermaG C Roberts
Nov 7, 2018·Journal of the American Chemical Society·Anil R MhashalDan Thomas Major

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