Role of arginine residue in saccharopine dehydrogenase (L-lysine-forming) from bakers' yeast

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Abstract
The baker''s yeast saccharopine dehydrogenase was inactivated by 2,3-butanedione following pseudo-1st-order reaction kinetics. The pseudo-1st-order rate constant for inactivation was linearly related to the butanedione concentration and a value of 7.5 M-1 min-1 was obtained for the 2nd-order rate constant at pH 8.0 and 25.degree. C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as 8 arginine residues were lost on prolonged incubation with butanedione, only 1 residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analog of substrate lysine, in the presence of NADH and .alpha.-ketoglutarate. Since leucine binds only to the enzyme.cntdot.NADH.cntdot..alpha.-ketoglutarate complex, an arginine residue located near the binding site for the amino acid substrate apparently is modified. Titration with leucine showed that the reaction of butanedione took place with the enzyme.cntdot.NADH.cntdot..alpha.-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. An arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.