Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.
Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.
复制标题
使用 PDC1-PDC6 融合蛋白研究半胱氨酸在啤酒酵母丙酮酸脱羧酶激活和失活中的作用。
DOI：
10.1021/bi00061a031
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Jordan,F
中科院分区：
文献类型：
--
作者：
Zeng,X;Farrenkopf,B;Hohmann,S;Dyda,F;Furey,W;Jordan,F
Revised Manuscript Received November 30, 1992 abstract: Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers’ yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdcl containing four cysteines/subunit (positions 69,152, 221, and 222) with that of a fusion enzyme (pdcl-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdcl and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme.(p-Nitrobenzoyl) formic acid is an excellent alternate substrate, whose decarboxylation product p-nitroben-zaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdcl, to (£)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdcl, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdcl with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F.(1991)/. Am. Chem. Soc. 113, 5842-49]. Of the two adjacent cysteines, Cys221 and Cys222, at least Cys221, and perhaps even Cys222, is likely to be important for fully functioning native pyruvate decarboxylase from brewers’ yeast.The simplest nonoxidative decarboxylation of a-keto acids is performed by pyruvate decarboxylase (PDC, 1 EC 4.1. 1.1). As is the case with nearly all such enzymes, thiamin diphosphate (ThDP) is an essential cofactor (see reviews in Krampitz (1969, 1970), Sable and Gubler (1982), Kluger (1987), Schellenberger and Schowen (1988), and Bisswanger and Ullrich (1991)). While the enzyme is capable of accelerating the reaction over the nonenzymic, ie, ThDP-catalyzed, model reaction by a factor of 1012 (Alvarez et al., 1991), chemicalmodification studies have not revealed the participation of any potential general acid-base or nucleophilic enzymic side chain in the reaction mechanism, except possibly for Cys (Ullrich, 1982). That Cys modification greatly reduces the activity has been reported repeatedly for nearly 50 years (see Jordan et al.(1988) for a detailed reference list). It has also been knownfor some time that theenzyme isolated from
