Probing the active site of flavocytochrome b2 by site-directed mutagenesis

The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution [Mathews & 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 degrees 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 Lys349, 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.