Jain M K, Rogers J, Hendrickson H S, Berg O G
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Biochemistry. 1993 Aug 17;32(32):8360-7. doi: 10.1021/bi00083a040.
The effect of detergents on the overall catalytic turnover by secreted phospholipase A2 (PLA2) on codispersions of the substrate phospholipid is characterized. The overall rate of interfacial catalytic turnover depends on the effective substrate "concentration" (mole fraction) that the bound enzyme "sees" at the interface. Therefore, besides the intrinsic catalytic turnover rate determined by the Michaelis-Menten cycle in the interface [Berg et al. (1991) Biochemistry 30, 7283], two other interfacial processes significantly alter the overall effective rate of hydrolysis: first, the fraction of the total enzyme at the interface; second, the rate of replenishment of the substrate. At low mole fractions (< 0.3), bile salts promote the binding of pig pancreatic PLA2 to zwitterionic vesicles, and the rate of hydrolysis increases with the fraction of the enzyme in the interface. At higher (> 0.3) mole fractions of the detergent, the bilayer is disrupted, and the rate of hydrolysis decreases by more than a factor of 10. The detergent-dependent decrease in the rate of hydrolysis of the sn-2-oxyphospholipids is much larger than that of sn-2-thiophospholipid, and therefore the element effect (O/S ratio) decreases from about 10 in bilayers to less than 2 in mixed micelles. This loss of the element effect in mixed micelles shows that the chemical step is no longer rate-limiting during the hydrolysis of mixed micelles formed by the disruption of vesicles by the detergent. Such effects were observed with phospholipase A2 from several sources acting on substrates dispersed in a variety of detergents including bile salts, 2-deoxylysophosphatidylcholine, and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了去污剂对分泌型磷脂酶A2（PLA2）催化底物磷脂共分散体的总体催化周转率的影响。界面催化周转的总体速率取决于结合酶在界面处“看到”的有效底物“浓度”（摩尔分数）。因此，除了由界面处的米氏循环确定的内在催化周转率[伯格等人（1991年）《生物化学》30，7283]外，另外两个界面过程也会显著改变总体有效水解速率：第一，界面处总酶的比例；第二，底物的补充速率。在低摩尔分数（<0.3）时，胆盐促进猪胰PLA2与两性离子囊泡的结合，水解速率随界面处酶的比例增加而增加。在去污剂的摩尔分数较高（>0.3）时，双层膜被破坏，水解速率降低超过10倍。去污剂依赖性的sn-2-氧磷脂水解速率降低比sn-2-硫代磷脂大得多，因此元素效应（O/S比）从双层膜中的约10降至混合胶束中的小于2。混合胶束中元素效应的这种损失表明，在去污剂破坏囊泡形成的混合胶束水解过程中，化学步骤不再是限速步骤。在几种来源的磷脂酶A2作用于分散在多种去污剂（包括胆盐、2-脱氧溶血磷脂酰胆碱和 Triton X-100）中的底物时观察到了这种效应。（摘要截断于250字）
