Protein Science, Vol 7, Issue 7 1531-1537, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

About the pK(a) of the active-site histidine in flavocytochrome b(2) (yeast L-lactate dehydrogenase)

K. S. RAO and F. LEDERER
Present address: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, Michigan 48201.

Flavocytochrome b(2) or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate {alpha}-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on N{epsilon} of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-(3)H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k(e)(H), for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (+/-0.03) M(-1) s(-1). Using the Eigen equation, this figure yields a pK(a) of 9.1 +/- 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291-295). The mechanistic significance of these results is discussed.
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