Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2.
Flavocytochrome b2 from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour of larger substrates, three mutant enzymes have been produced: A198G, L230A and the double mutant A198G/L230A. As a means of quantifying the overall kinetic effect of a mutation, substrate-specificity profiles were produced from steady-state experiments with (S)-2-hydroxy acids of increasing chain length, through which the catalytic efficiency of each mutant enzyme with each substrate could be compared with the corresponding wild-type efficiency. The Ala-198-->Gly mutation had little influence on substrate specificity and caused a general decrease in enzyme efficiency. However, the Leu-230-->Ala mutation caused the selectivity for 2-hydroxyoctanoate over lactate to increase by a factor of 80.
Daff
S
S
Edinburgh Centre for Molecular Recognition, Department of Chemistry, University of Edinburgh, Scotland, UK.
Manson
F D
FD
Reid
G A
GA
Chapman
S K
SK
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005998:Glycine; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D007930:Leucine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007773:Lactates; D019344:Lactic Acid; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D007930:Leucine; D000409:Alanine; D005998:Glycine