Yeast inorganic pyrophosphatase. A model for active-site structure based on 113Cd2+ and 31P NMR studies.
Yeast inorganic pyrophosphatase. A model for active-site structure based on 113Cd2+ and 31P NMR studies.
复制标题
酵母无机焦磷酸酶。
DOI：
10.1021/bi00316a019
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Cooperman,BS
中科院分区：
文献类型：
--
作者：
Welsh,KM;Cooperman,BS
凯瑟琳M. Welsh* 和巴里S. Cooperman* 摘要：平衡透析和~（113）Cd ~（2+）NMR研究表明，在酵母无机焦磷酸酶亚基上存在四个明确的Cd ~（2+）位点。平行的~（31）P NMR研究证实了每个亚基上存在两个P ~+结合位点，并为少量的酶结合的无机焦磷酸盐与酶结合的P ~+平衡提供了强有力的确证性证据。这种无机焦磷酸盐的形成是通过早期的化学分析证明的[Welsh，K. M.，阿米蒂奇岛M.，& Cooperman，B. S.（1983）Biochemistry 22，1046-1054]。在这篇相同的较早的论文中，我们提供了关于在无机焦磷酸酶（PPase），1 EC 3.6之间的内球接触的证据。1.1，是许多磷酰基转移酶的典型特征，其活性需要二价金属离子。近年来，已经花费了相当大的努力来确定每个亚基的这种离子的数量[PPase伊萨相同亚基的二聚体，285个氨基酸残基长（Heinrikson等人，1973; Cohen等人，1978）]的活性和相对位置的酶表面上的这些离子，相对于彼此和结合底物所需的。虽然Mg 2+赋予无机焦磷酸盐（PP）或无机磷酸盐（P）（在H2O-P氧交换中）作为底物的最高催化活性，但Zn 2+、Mn 2+和Co 2+也赋予实质性活性（Kunitz，1952; Welsh等人，1983年b）。通过使用平衡透析或金属离子电极测量，对Mg 2+、Mn 2+和Co2+直接结合的详细研究（Rapoport等人，1973; Cooperman等人，1981），和间接地，通过测量作为二价金属离子浓度的函数的PPase的功能性质（莫伊& Butler，1972; Springs et al.，1981年; Knight等人，1984）得出的结论是，活性酶每个亚基需要三个二价金属离子，此外，每个亚基还可以结合第四个二价金属离子，该第四个二价金属离子没有明确的催化功能。对Mn 2 +-酶的EPR研究提供了在活性位点附近存在三种二价金属离子的证据（Banerjee & Cooperman，1983; Knight等人，1984），而~（31）P NMR实验表明，在两个P位点的高亲和力处，酶结合的金属离子与P之间存在内球相互作用，而在两个P位点的低亲和力处，酶结合的金属离子与P之间仅存在外球相互作用（哈姆&库珀曼，1978; Welsh等，1983年a）。
Katherine M. Welsh* and Barry S. Cooperman* abstract: Equilibrium dialysis and 113Cd2+ NMR studies in the presence of inorganic phosphate (P¡) provide clear ev-idence for the existence of four well-defined Cd2+ sites per yeast inorganic pyrophosphatase subunit. Parallel 31P NMR studies demonstrate the existence of two binding sites per subunit for P¡ and provide strong confirmatory evidence for a small amount of enzyme-bound inorganic pyrophosphate in equilibrium with enzyme-bound P¡. Such inorganic pyro-phosphate formation was demonstrated by chemical analysis earlier [Welsh, K. M., Armitage, I. M., & Cooperman, B. S.(1983) Biochemistry 22, 1046-1054], In this same earlier paper, we provided evidence for inner-sphere contact between^ east inorganic pyrophosphatase (PPase), 1 EC 3.6. 1.1, is typical of many phosphoryl transfer enzymes in requiring divalent metal ions for activity. In recent years, a considerable effort has been expended todetermine both the number of such ions per subunit [PPase isa dimer of identical subunits, 285 amino acid residues long (Heinrikson et al., 1973; Cohen et al., 1978)] required for activity and the relative placement of such ions on the enzyme surface, with respect both to each other and to bound substrate. Although Mg2+ confers the highest catalytic activity with inorganic pyrophosphate (PP¡) or inorganic phosphate (P¡)(in H20-P¡ oxygen exchange) as substrate, Zn2+, Mn2+, and Co2+ also confer substantial activity (Kunitz, 1952; Welsh et al., 1983b). Detailed studies of Mg2+, Mn2+, and Co2+ binding, both directly, by using equilibrium dialysis or metal ion electrode measurements (Rapoport et al., 1973; Cooperman et al., 1981), and indirectly, by measuring functional properties of PPase asa function of divalent metal ion concentration (Moe & Butler, 1972; Springs et al., 1981; Knight et al., 1984), have led to the conclusion that active enzyme requires three divalent metal ions per subunit and that, in addition, a fourth divalent metalion, having no clear cat-alytic function, may also be bound per subunit. EPR studies on the Mn2+-enzyme have provided evidence for the presence of three divalent metal ions in some proximity at the active site (Banerjee & Cooperman, 1983; Knight et al., 1984), and 31P NMR experiments have indicated an inner-sphere interaction between enzyme-bound metal ion and P¡ bound in the higher affinity of the two P¡ sites but only an outer-sphere interaction between enzyme-bound metal ion and P¡ bound in the lower affinity of the two P¡ sites (Hamm & Cooperman, 1978; Welsh et al., 1983a).