IDENTIFICATION OF GLUTAMATE-344 AS THE CATALYTIC RESIDUE IN THE ACTIVE-SITE OF PIG-HEART COA TRANSFERASE

The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transfe rase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the met abolism of ketone bodies in the mammalian mitochondrion. It is known t hat its catalytic mechanism involves the transient thioesterification of an active-site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous a utolytic fragmentation that occurs at the active site when the enzyme- CoA covalent intermediate is heated. The presence of protease inhibito rs has no effect on the extent of cleavage detectable by SDS-PAGE, sup porting the view that this fragmentation is indeed autolytic. This fra gmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the r esulting C-terminal fragment is blocked at its N-terminus by a pyroglu tamate moiety, it is not amenable to direct sequencing by the Edman de gradation method. As an alternative, we have studied a peptide (peptid e D) generated specifically by autolysis of the nicked enzyme and pred icted to have an N-terminus corresponding to the site of proteolysis a nd a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by elec trospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conser ved in all sequenced CoA transferases, is the catalytically active ami no acid. This information should prove useful to future mutagenesis wo rk aimed at better understanding the active-site structure and catalyt ic mechanism of CoA transferase.