The comparative enzymology of lactic dehydrogenases. IV. Function of sulfhydryl groups in lactic dehydrogenases and the sequence around the essential group.

Hq lactic dehydrogenases crystallized from heart and Mq lactic dehydrogenases crystallized from muscle tissues of various vertebrates have four functionally essential thiol groups per molecule of enzyme (1). This value suggests that there is one active site thiol group for each of the four subunits of these lactic dehydrogenases. Attempts to introduce a noninterchangeable thiol reagent specifically at the active site of any of these enzymes in the native configuration have failed. Gold and Segal (2) have succeeded in introducing 4 eq of N , N(dimethylamino-3,5dinitrophenyl)maleimide into beef Hh lactic dehydrogenasel by freezing and thawing the enzyme and the reagent in 0.25 M sodium chloride. The enzyme so labeled showed an 80% reduction in activity. The extent of this inactivation was reduced in the presence of NADH. The other papers in this series (3, 7)2 outline the preparation of crystalline lactic dehydrogenases from a variety of mammals, birds, reptiles, fishes, an amphibian, a cyclostome, and a crustacean. We have determined the total thiol content of these various Hq and Mr lactic dehydrogenases by titration with phydroxymercuribenzoate in urea. In addition, we have determined the effect of p-hydroxymercuribenzoate-binding on enzymatic activity as well as the influence of coenzyme on the p-hydrosymercuribenzoate-induced inactivation. This survey has led to the finding that the Mq lactic dehydrogenase from bullfrog (Rana catesbeiana) has only two other thiol groups in addition to the four essential ones. The nonessential thiols have been blocked by iodoacetate in the native enzyme, thus permitting identification of the essential thiols by alkylation with radioactive iodoacetate in urea. Preliminary reports of this work have been delivered (8, 9).