Lantum Hoffman B M, Liebler Daniel C, Board Philip G, Anders M W
Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, New York 14642, USA.
Chem Res Toxicol. 2002 May;15(5):707-16. doi: 10.1021/tx025503s.
Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.
谷胱甘肽转移酶ζ（GSTZ1-1）分别催化马来酰乙酰乙酸或马来酰丙酮（MA）顺反异构化为富马酰乙酰乙酸或富马酰丙酮（FA）。GSTZ1-1还催化一系列α-卤代酸（包括二氯乙酸）的谷胱甘肽依赖性生物转化。本研究的目的是研究MA和FA使hGSTZ1-1失活的机制，并确定在有和没有谷胱甘肽存在的情况下MA和FA对hGSTZ1-1的共价修饰。MA和FA（0.01-1 mM）以浓度和时间依赖性方式使所有hGSTZ1-1多态性变体失活，并且这种失活被谷胱甘肽阻断。hGSTZ1c-1c的C16A突变体被MA和FA部分失活。对hGSTZ1多态性变体的胰蛋白酶消化物进行电喷雾电离串联质谱分析和SALSA（光谱分析评分算法）分析表明，hGSTZ1-1的活性位点（SSCSWR）和C末端（LLVLEAFQVSHPCR）半胱氨酸残基被MA和FA共价修饰。MA和FA加合导致修饰肽离子及其MS-MS碎片离子的质量发生156-Da的诊断性位移。当hGSTZ1-1多态性变体在S-甲基谷胱甘肽存在下与MA或FA孵育时，活性位点半胱氨酸残基的烷基化（而非C末端半胱氨酸的烷基化）相对较弱。这些数据表明，MA和FA是hGSTZ1-1的底物和产物失活剂，并且在没有谷胱甘肽的情况下在活性位点半胱氨酸残基处共价修饰hGSTZ1-1。谷胱甘肽阻断失活的观察结果表明，谷胱甘肽与活性位点的结合可防止MA或FA与活性位点半胱氨酸残基反应。这些数据还表明，MA和FA可能共价修饰并使其他具有可及半胱氨酸残基的蛋白质失活，从而可能导致二氯乙酸诱导的毒性或与I型高酪氨酸血症相关的毒性。
