ATYPICAL VELOCITY RESPONSE BY PYRUVATE-CARBOXYLASE TO INCREASING CONCENTRATIONS OF ACETYL-COENZYME-A

The interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, was investigated. The velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were dependent on the fixed pyruvate concentration used in the assay solution. By varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme-carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalyzing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.