Leucine dehydrogenase of Bacillus sphaericus: Sulfhydryl groups and catalytic sites.

Abstract

Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl2, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl2 was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group.