Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.
Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.
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大鼠肾高分子量磷脂酶 A2 的动力学分析：二价金属依赖性酶在含有产物的囊泡上的捕获。
DOI：
10.1021/bi00130a012
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Gelb,MH
中科院分区：
文献类型：
--
作者：
Ghomashchi,F;Schüttel,S;Jain,MK;Gelb,MH
华盛顿大学化学和生物化学系，西雅图，华盛顿98195，和特拉华州大学化学和生物化学系，纽瓦克，特拉华州19716接收日期：1991年10月30日;修订版手册接收日期：1992年1月24日摘要：大鼠肾脏高分子量磷脂酶A2催化1-棕榈酰-2-花生四烯酸-sn-甘油-3-磷酸胆碱囊泡水解动力学表现出异常行为。反应过程持续几分钟，然后在仅5-10%的可用底物被水解后停止。加入更多的酶而不是更多的底物导致新一轮的水解。虽然这一发现最初表明，在周转过程中，这种酶会失活，但这一结论无法得到证实。在反应停止后，向反应中加入含有0.15 M NaCl和牛血清白蛋白的缓冲液，导致脂解的重新开始。酶不被反应产物强烈抑制。虽然该酶不可逆地结合到由纯的1-棕榈酰-2-花生四烯酰-5n-甘油-3-磷酸胆碱组成的囊泡，但它确实不可逆地被捕获在含有临界摩尔百分比的反应产物的囊泡上。这种捕获是反应停止的最可能的解释。酶与1，2-二棕榈酰-sn-甘油-3-磷酸胆碱囊泡的结合和这些囊泡中所含的1-硬脂酰-2-[3 H]花生四烯酰-sn-甘油磷酸胆碱的水解都需要产物的存在。此外，酶的捕获与催化周转无关。捕获对囊泡中存在的脂肪酸的结构敏感，并且需要二价金属（Ca 2+、Sr 2+、Ba 2+或Mg 2+）的存在。由于酶活性所需的金属浓度与促进捕获所需的量相关，因此建议金属的作用仅是促进酶的界面结合。
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, and Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Received October 30, 1991; Revised Manuscript Received January 24, 1992 abstract: The kinetics of hydrolysis of l-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that theenzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bindirreversibly to vesicles composed of pure l-palmitoyl-2-arachidonyl-5 «-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to l, 2-dipalmitoyl-j «-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H] arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitiveto the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.