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 transferase [3-oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that 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 autolytic fragmentation that occurs at the active site when the enzyme-CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS-PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C-terminal fragment is blocked at its N-terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N-terminus corresponding to the site of proteolysis and a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active-site structure and catalytic mechanism of CoA transferase.
Rochet
J C
JC
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Bridger
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D015256:Coenzyme A-Transferases / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004951:Esterification; D005971:Glutamates; D018698:Glutamic Acid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D005971:Glutamates; D010446:Peptide Fragments; D018698:Glutamic Acid; D015256:Coenzyme A-Transferases; C021460:3-ketoacid CoA-transferase