L-mandelate dehydrogenase from Rhodotorula graminis: comparisons with the L-lactate dehydrogenase (flavocytochrome b2) from Saccharomyces cerevisiae.
L-Lactate dehydrogenase (L-LDH) from Saccharomyces cerevisiae and L-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis are both flavocytochromes b2. The kinetic properties of these enzymes have been compared using steady-state kinetic methods. The most striking difference between the two enzymes is found by comparing their substrate specificities. L-LDH and L-MDH have mutually exclusive primary substrates, i.e. the substrate for one enzyme is a potent competitive inhibitor for the other. Molecular-modelling studies on the known three-dimensional structure of S. cerevisiae L-LDH suggest that this enzyme is unable to catalyse the oxidation of L-mandelate because productive binding is impeded by steric interference, particularly between the side chain of Leu-230 and the phenyl ring of mandelate. Another major difference between L-LDH and L-MDH lies in the rate-determining step. For S. cerevisiae L-LDH, the major rate-determining step is proton abstraction at C-2 of lactate, as previously shown by the 2H kinetic-isotope effect. However, in R. graminis L-MDH the kinetic-isotope effect seen with DL-[2-2H]mandelate is only 1.1 +/- 0.1, clearly showing that proton abstraction at C-2 of mandelate is not rate-limiting. The fact that the rate-determining step is different indicates that the transition states in each of these enzymes must also be different.
Smékal
O
O
Department of Chemistry, University of Edinburgh, U.K.
Yasin
M
M
Fewson
C A
CA
Reid
G A
GA
Chapman
S K
SK
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism / Q000494:pharmacology; D019344:Lactic Acid; D008333:Mandelic Acids / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D012248:Rhodotorula / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D007773:Lactates; D008333:Mandelic Acids; D019344:Lactic Acid; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase; C021538:L-mandelate dehydrogenase; C037938:mandelic acid