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-Isopropylmalate Synthase from Salmonella typhimurium

ANALYSIS OF THE QUATERNARY STRUCTURE AND ITS RELATION TO FUNCTION

From the ¹ From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Sedimentation equilibrium centrifugation and intraoligomer cross-linking were used to further investigate the quaternary structure of -isopropylmalate synthase.

Analytical ultracentrifugation of enzyme denatured in 6 m guanidine hydrochloride and 0.1 m 2-mercaptoethanol yielded a weight-average molecular weight for the polypeptide chain between 47,000 and 52,000, depending on the partial specific volume employed. This result is in good agreement with the value of approximately 50,000 established previously by gel electrophoresis in sodium dodecyl sulfate. Gel electrophoresis in the presence of 5 m urea resulted in one protein band, further supporting the concept that the subunits are similar if not identical.

Analysis of the association-dissociation equilibrium of the native enzyme by sedimentation equilibrium ultracentrifugation led to the conclusion that the system can be adequately described as an ideal monomer-tetramer equilibrium with an association constant, K₄ = [A₄]/[A]⁴, of 9 x 10¹⁶ m^-3. Neither substrates, employed individually, nor products were found to have a significant effect on the equilibrium. However, when enzyme which had been reacted with the potential active site-labeling reagent bromopyruvate was analyzed in the presence of acetyl-CoA, the associated form of the enzyme was stabilized. Thus, taking into account the dissociative effect of leucine shown earlier, the substrates and the feedback inhibitor appear to influence the association-dissociation equilibrium in opposite directions.

An enzymatically active, nondissociable tetrameric form of the enzyme was isolated by gel filtration following intraoligomer cross-linking with dimethyl suberimidate in the presence of acetyl-CoA and -ketoisocaproate. This form of the enzyme was largely desensitized against inhibition by leucine. The potential of the cross-linking method to isolate and characterize enzyme species of a defined state of aggregation, otherwise practically inaccessible because of rapid association-dissociation equilibrium, is indicated.

The possible significance of the association-dissociation equilibrium for catalysis and regulation of -isopropylmalate synthase is discussed.

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