单卤代丙氨酸和多卤代丙氨酸对磷酸吡哆醛依赖性酶的失活作用。

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines.

作者信息

Silverman R B, Abeles R H

出版信息

Biochemistry. 1976 Oct 19;15(21):4718-23. doi: 10.1021/bi00666a028.

Abstract

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-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 gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-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 HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta 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 gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.

摘要

β,β-二氯丙氨酸和β,β,β-三氟丙氨酸可使多种依赖磷酸吡哆醛的酶不可逆地失活，这些酶催化β-或γ-消除反应。失活过程与时间相关，且失活速率与酶浓度呈一级关系。这表明失活是由于多卤代丙氨酸在活性位点产生的某种物质对酶进行共价修饰所致（即自杀失活）。单卤代丙氨酸是底物，不会导致失活。对于γ-胱硫醚酶，已证明[1-¹⁴C]β,β,β-三氟丙氨酸会发生共价且化学计量的结合。有人提出失活机制涉及失活剂与酶结合的吡哆醛之间形成席夫碱，随后二氯丙氨酸中的HCl或三氟丙氨酸中的HF被消除。这会导致形成β-卤代-α,β-不饱和亚胺，一种活化的迈克尔受体。活性位点处亲核试剂的迈克尔加成导致酶的共价标记和失活。丙氨酸消旋酶也会被这两种多卤代丙氨酸失活。谷氨酸-丙酮酸转氨酶和谷氨酸-草酰乙酸转氨酶会被单卤代丙氨酸失活，但不会被多卤代丙氨酸失活。