STRATEGIC MANIPULATION OF THE SUBSTRATE-SPECIFICITY OF SACCHAROMYCES-CEREVISIAE FLAVOCYTOCHROME B(2)

Flavocytochrome b(2) from Saccharomyces cerevisiae acts physiologicall y as an L-lactate dehydrogenase. Although L-lactate is its primary sub strate, the enzyme is also able to utilize a variety of other (S)-2-hy droxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues requ ired for catalysis. However, the residues Ala-198 and Leu-230, found i n the X-ray-crystal structure to be in contact with the substrate meth yl group, are not well conserved. We propose that the interaction betw een these residues and a prospective substrate molecule has a signific ant effect on the substrate specificity of the enzyme. In an attempt t o modify the specificity in favour of larger substrates, three mutant enzymes have been produced: A198G, L230A and the double mutant A198G/L 230A. As a means of quantifying the overall kinetic effect of a mutati on, substrate-specificity profiles were produced from steady-state exp eriments with (S)-2-hydroxy acids of increasing chain length, through which the catalytic efficiency of each mutant enzyme with each substra te could be compared with the corresponding wild-type efficiency. The Ala-198-->Gly mutation had little influence on substrate specificity a nd caused a general decrease in enzyme efficiency. However, the Leu-23 0-->Ala mutation caused the selectivity for 2-hydroxyoctanoate over la ctate to increase by a factor of 80.