Alpha-Isopropylmalate synthase from Alcaligenes eutrophus H 16. II. Substrate specificity and kinetics.

Abstract

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following alpha-keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): alpha-ketoisovalerate, alpha-keto-n-valerate, alpha-ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, valeryl-CoA, and crotonyl0CoA. alpha-Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct L-leucine. The substrate saturation curves of alpha-ketoisovalerate or other alpha-keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between nH-values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (alpha-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 micronM) inactivated the enzyme irreversibly. The 3'-phosphate of coenzyme A and the free carboxyl group of alpha-ketoisovalerate were involved in optimal binding of these substrates, but 3'-dephospho-acetyl-coenzyme A and the methylester of alpha-keto-isovalerate were also converted by this enzyme. A CH3--CH2-grouping of the alpha-keto acids seemed to be necessary for binding this substrate.