Inactivation of Pyridoxal Phosphate Dependent Enzymes by Mono- and Polyhaloalanines

Abstract

β,β-Dichloro- and.β,β,β-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze β- or γ-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For γ-cystathionase, covalent and stoichiometric attachment of [1-14C]β,β,β-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HCl from dichloroalanine or HF from trifluoroalanine. This results in the formation of a β-halo-α,β unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and glutamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines. © 1976, American Chemical Society. All rights reserved.