Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine.
The pyridoxal form of both cytosolic and mitochondrial aspartate aminotransferase is irreversibly inactivated consequent to its interaction with the beta,gamma-unsaturated substrate analogue vinylglycine. Per catalytic cycle, 90% of the enzyme molecules are inactivated while 10% escape inactivation by transamination to the pyridoxamine form. In the presence of vinylglycine plus 2-oxoglutarate, inactivation is complete because of retransamination of the pyridoxamine form to the susceptible pyridoxal form. Peptide analyses after inactivation with [1-14C]vinylglycine showed that vinylglycine alkylates the active-site lysine residue 258 which forms the internal aldimine with the coenzyme pyridoxal 5'-phosphate. The coenzyme itself is left intact; resolution of the inactivated enzyme by base or trichloroacetic acid yields pyridoxal-5'-P. The absorption spectrum of the inactivated enzyme (lambdamax 335 nm) suggests that the cofactor is bound as a substituted aldimine. The proposed pathway of alkylation of Lys-258 involves abstraction of the alpha proton from vinylglycine, isomerization to the alpha,beta-unsaturated enamine, and subsequent nucleophilic attack of the epsilon-amino group of the lysyl residue at the beta carbon of the inhibitor. The determination of the amino acid sequence around the coenzyme-binding lysyl residue in the mitochondrial isoenzyme from chicken gave Ala-(epsilon-Pxy)Lys-Asn-Met-(Gly,Leu,Tyr) which is identical with the other mitochondrial transaminases examined so far.
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D005998:Glycine / Q000031:analogs & derivatives*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011730:Pyridoxal; D013552:Swine
D007527:Isoenzymes; D010446:Peptide Fragments; D011730:Pyridoxal; C006507:vinylglycine; D001219:Aspartate Aminotransferases; D005998:Glycine