ESSENTIAL ARGININE RESIDUES IN LACTATE-DEHYDROGENASE FROM GERMINATINGSOYBEAN

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromalography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arg inine residues in the mechanism of enzyme action was determined throug h kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). Th e pK values found were 12.4 for the enzyme-substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione and p-hydroxyphcnylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occ urs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyox al was determined. It appears there are at least two arginine residues in the active site of the enzyme.