L(-MANDELATE DEHYDROGENASE FROM RHODOTORULA-GRAMINIS - PURIFICATION, PARTIAL CHARACTERIZATION AND IDENTIFICATION AS A FLAVOCYTOCHROME-B())

L(+)-Mandelate dehydrogenase was purified to homogeneity from the yeas t Rhodotorula graminis KGX 39 by a combination of (NH4)2SO4 fractionat ion, ion-exchange and hydrophobic-interaction chromatography and gel f iltration. The amino-acid composition and the N-terminal sequence of t he enzyme were determined. Comprehensive details of the sequence deter minations have been deposited as Supplementary Publication SUP 50172 ( 4 pages) at the British Library Document Supply Centre, Boston Spa, We therby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtaine d on the terms indicated in Biochem. J. (1993) 289, 9. The enzyme is a tetramer as judged by comparison of its subunit M(r) value of 59 100 and native M(r) of 239900, estimated by SDS/PAGE and gel filtration re spectively. There is one molecule of haem and approx. one molecule of non-covalently bound FMN per subunit. 2,6-Dichloroindophenol, cytochro me c and ferricyanide can all serve as electron acceptors. L(+)-Mandel ate dehydrogenase is stereospecific for its substrate. D(-)-Mandelate and L(+)-hexahydromandelate are competitive inhibitors. The enzyme has maximum activity at pH 7.9 and it has a pI value of 4.4. HgCl2 and 4- chloromercuribenzoate are potent inhibitors, but there is no evidence that the enzyme is subject to feedback inhibition by potential metabol ic effectors. The evidence suggests that L(+)-mandelate dehydrogenase from R. graminis is a flavocytochrome b which is very similar to, and probably (at least so far as the haem domain is concerned) homologous with, certain well-characterized yeast L(+)-lactate dehydrogenases, an d that the chief difference between them is their mutually exclusive s ubstrate specificities.