THIOLTRANSFERASE IN HUMAN RED-BLOOD-CELLS - KINETICS AND EQUILIBRIUM
THIOLTRANSFERASE IN HUMAN RED-BLOOD-CELLS - KINETICS AND EQUILIBRIUM
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DOI：
10.1021/bi00100a023
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发表时间：
1991-09-10
期刊：
影响因子：
2.9
通讯作者：
HOCEVAR, BA
中科院分区：
文献类型：
--
作者：
MIEYAL, JJ;STARKE, DW;HOCEVAR, BA
来自人红细胞的巯基转移酶（HRBC TTase）与GSSG还原酶偶联，催化原型底物羟乙基二硫化物（HEDS）和S-磺基半胱氨酸钠以及其他同型和杂二硫化物（包括蛋白质混合二硫化物白蛋白-S-S-半胱氨酸）的谷胱甘肽（GSH）依赖性还原。 尽管底物的表观K（M）值在20倍以上的范围内变化，但V（max）值非常接近，通常小于2倍，表明氧化底物与酶的初始相互作用不是速率决定性的。 HRBC TTase被碘乙酰胺（IAA）灭活，这是通过用二硫化物预处理来防止的。 IAA失活的pH依赖性给出了非常低的表观pK（a）3.5，其与离子强度（0.05-2 M）无关。 在pH 6时，用[C-14]IAA处理后，一个放射性标记的羧酰胺甲基部分与酶结合。 这种不寻常的巯基反应性表明，TTase的活性位点半胱氨酸部分可能参与与羧酸根部分的氢键。 与此相反，二硫键还原的GSH依赖性TTase催化的pH依赖性显示在pH 8.0附近的拐点，也表明氧化底物与活性位点硫醇的初始反应不参与速率测定。 HRBC TTase和大鼠肝TTase的两种底物动力学研究（例如，[GSH]和[HEDS]独立变化）在双倒数图（1/上升vs 1/S）上给出交叉线图案，表明TTase反应的顺序机制，而不是乒乓机制。 因此，GSH和二硫化物底物似乎在二硫化物还原产物释放之前与还原酶相互作用，而不是GSH仅仅作为氧化酶的还原剂。 当省略GSSG还原酶时，显示TTase催化从任一组底物开始的相同平衡位置的方法（例如，GSH + HEDS对比GSSG + β-巯基乙醇）。 因此，TTase的作用是完全可逆的，催化的方向和反应的程度取决于各自底物的相对浓度和GSSG还原酶的偶联作用。
Thioltransferase from human red blood cells (HRBC TTase), couled to GSSG reductase, catalyzed glutathione (GSH)-dependent reduction of prototype substrates hydroxyethyl disulfide (HEDS) and sodium S-sulfocysteine as well as of other homo- and heterodisulfides, including the protein mixed disulfide albumin-S-S-cysteine. Whereas apparent K(M) values for the substrates varied over more than a 20-fold range, the V(max) values agreed quite closely, usually within less than a factor of 2, suggesting that initial interaction of oxidized substrate with enzyme is not rate determining. HRBC TTase was inactivated by iodoacetamide (IAA), and this was prevented by pretreatment with disulfides. The pH dependence of IAA inactivation gave a remarkably low apparent pK(a) of 3.5, which was independent of ionic strength (0.05-2 M). At pH 6, one radiolabeled carboxyamidomethyl moiety was bound to the enzyme after treatment with [C-14]IAA. This unusual thiol reactivity suggests that the active-site cysteine moiety of the TTase may be involved in a hydrogen bond with a carboxylate moiety. In contrast, the pH dependence for GSH-dependent TTase catalysis of disulfide reduction displayed an inflection point near pH 8.0, also suggesting that the initial reaction of oxidized substrate with the active-site thiol is not involved in rate determination. Two substrate kinetic studies of HRBC TTase and rat liver TTase (e.g., [GSH] and [HEDS] varied independently) gave patterns of intersecting lines on double-reciprocal plots (1/upsilon vs 1/S), indicating a sequential mechanism for the TTase reactions, rather than a ping-pong mechanism. Thus, both GSH and the disulfide substrate appear to interact with the reduced enzyme before the products of disulfide reduction are released, rather than GSH acting simply as a reductant of the oxidized enzyme. When GSSG reductase was omitted, the TTase was shown to catalyze the approach to the same equilibrium position starting from either set of substrates (e.g., GSH + HEDS vs GSSG + beta-mercaptoethanol). Thus, TTase action is fully reversible, and the direction of catalysis and extent of reaction depend on the relative concentrations of the respective substrates and on the coupled action of GSSG reductase.