Affinity labelling of rat liver acetyl-CoA car☐ylase by a 2′,3′-dialdehyde derivative of ATP

Abstract

The interaction of rat liver acetyl-CoA car☐ylase with a 2′,3′-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3− accelerated the process. Ki values for oATP in the absence and presence of HCO3− were 0.35 ± 0.04 and 0.5 ± 0.06 mM , and those of the modification constant (kmod) were 0.11 and 0.26 min−1 respectively. oATP completely inhibited the [14C]ADP ⇌ ATP exchange and did not effect the [14C]acetyl-CoA ⇌ malonyl-CoA exchange. Incorporation of ∼1 equivalent of [3H]oATP per acetyl-CoA car☐ylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or β-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA car☐ylase by oATP. It was shown that the reagent apparently interacted selectively with the ɛ-amino group of lysine in the ATP-binding site to form a morpholine-like structure.