Affinity Labeling of the Active Center of l-Aspartate-β-decarboxylase with β-Chloro-l-alanine

Abstract

Abstract Interaction of β-chloro-l-[14C]alanine with l-aspartate-β-decarboxylase from Alcaligenes faecalis leads to inactivation associated with covalent binding of close to 1 mole of the 3-carbon chain of the analog per mole of active site. Evidence was obtained for two different types of binding. Thus, dialysis of the labeled enzyme against 0.05 m acetate (pH 6) led to loss of about half of the vitamin B6 cofactor without loss of 14C from the enzyme; before and after dialysis, about half of the 14C was released from the carboxyl group of the analog by treatment with ceric sulfate indicating the presence of a bound pyruvate residue. After dialysis of the labeled enzyme against 1 m acetate buffer (pH 5) containing 0.1 m l-glutamate, little additional vitamin B6 was lost (although similar dialysis of the holoenzyme releases all of the cofactor), but about half of the bound 14C was released; the 14C that remained bound to the dialyzed enzyme was not readily released by treatment with ceric sulfate. No evidence for nonidentical subunits was found. The data suggest that the two observed types of 14C-binding result from the aldimine-ketimine equilibrium; hydrolysis of the ketimine followed by loss of pyridoxamine 5'-phosphate at some sites competes with stabilization of the aldimine form by further interaction at other sites. The 14C-labeled enzyme was treated with cyanogen bromide and a labeled peptide was isolated. The label was released as β-hydroxy[14C]pyruvate from the peptide by treatment with mild alkali, 6 n HCl (105°, 18 hours), or on prolonged enzymatic digestion. Ammonolysis of the labeled peptide led to formation of glutamine and release of the label. The data indicate that the labeled derivative is β-hydroxypyruvate bound by ester linkage to a glutamic acid residue at the active center of the enzyme.