Probing the active site of flavocytochrome b2 by site‐directed mutagenesis
- 1 December 1988
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 178 (2), 329-333
- https://doi.org/10.1111/j.1432-1033.1988.tb14454.x
Abstract
The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers'' yeast (Saccharomyces cervisiae) has recently been solved at 0.24-nm resolution [Mathews and Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123-131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene. The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 .+-. 3 s-1 (25.degree.C, pH 7.5) to 4.3 .+-. 0.1 s-1. This substitution had, however, no discernable effect on Km for lactate (0.54 .+-. 0.03 mM for the mutant compared with 0.49 .+-. 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys340, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-1 to 160 s-1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of L-lactate oxidation.This publication has 27 references indexed in Scilit:
- A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenaseBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1987
- On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substratesEuropean Journal of Biochemistry, 1985
- Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectorsGene, 1985
- Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (l-lactate cytochrome c reductase) upon binding of pyruvate, the reaction productBiochimie, 1984
- [12] One-step gene disruption in yeastMethods in Enzymology, 1983
- Study of a Zone Highly Sensitive to Proteases in Flavocytochrome b2 from Saccharomyces cerevisiaeEuropean Journal of Biochemistry, 1981
- Structure of the active ternary complex of pig heart lactate dehydrogenase with S-lac-NAD at 2.7 Å resolutionJournal of Molecular Biology, 1981
- Microprocessor-controlled system for automatic acquisition of potentiometric data and their non-linear least-squares fit in equilibrium studiesTalanta, 1980
- Transformation in yeast: Development of a hybrid cloning vector and isolation of the can1 geneGene, 1979
- Detection of specific sequences among DNA fragments separated by gel electrophoresisJournal of Molecular Biology, 1975