Regulatory properties of acetokinase from Veillonella alcalescens

Acetokinase from Veillonella alcalescens catalyzes the virtually irreversible synthesis of adenosine triphosphate from acetyl phosphate and adenosine diphosphate. Kinetic analysis revealed that the enzyme was activated by acetyl phosphate and inhibited by adenosine triphosphate. Velocity curves obtained with increasing amounts of adenosine diphosphate were of the Michaelis-Menten type (rectangular hyperbolas) under all conditions employed. However, velocity curves generated by varying the level of acetyl phosphate were sigmoidal, suggesting homotropic interactions for this substrate. Alteration of the pH of the reaction mixture from 7.4 to 10 or addition of a substrate analogue, propionyl phosphate, resulted in the loss of cooperativity and changed the values of the Hill coefficient from 3 to 1. Data obtained in experiments with propionyl phosphate suggested the existence of separate effector sites on the enzyme. The inhibition curves for adenosine triphosphate were also sigmoidal with Hill coefficients ranging between 1 and 3, depending on the concentration of acetyl phosphate. Sedimentation studies with the partially purified enzyme indicated a polymeric structure with a maximum molecular weight of about 90,000 daltons; a subunit of approximately 29,000 daltons was also detected.