Molecular Theory of the Catalytic Function of Phospholipase A2

1.在饱和Ca~(2+)浓度下，磷脂酶A_2催化单分散和胶束底物的水解动力学表明，催化基团His 48的去质子化状态和酪氨酸52的质子化状态是催化反应所必需的。对于I组酶，活性部位N-末端α-氨基的电离状态的重要性也被指出。在与胶束底物结合时，酶-Ca^&lt；2+&gt；络合物的His 48和Tyr 52的pK值均显著向碱性侧移动，而与单分散底物的结合没有明显的pk移动。动力学研究表明，Ca~(2+)和~(2+)与两种类型的磷脂酶A_2S的结合是催化反应所必需的。与第一组磷脂酶A_2S的底物结合不依赖于与钙离子的结合，而与第二组酶的底物结合则促进了…超过10倍的钙^&lt；2+；狂欢。后者的结果与以下假设是一致的，即通过结合的Ca^&lt；2+&gt；离子与底物分子sn-2位的磷酰基和羰基配位来稳定中间络合物。然而，第一组酶的前一种结果似乎与这一机制不相容。对具有酰胺键而不是sn-2酯键的底物类似物的结合进行的X-射线结晶学研究表明，羰基氧和磷酰基部分直接与底物结合部位的结合钙离子相互作用，表明该类似物的结合应依赖于钙离子的结合。我们在溶液中的研究证实了这一点，该类似物对这两种酶的结合常数随着Ca^&lt；2+&gt；结合度的增加而显著增加，表明酶-底物类似物复合体和酶-类似物复合体在溶液中的结构与晶体中的结构非常相似，酶-类似物复合体的结构是由Ca^&lt；2+&gt；结合所稳定的。这两种类型的PLA2的酶-Ca^&lt；2+&gt；-真底物络合物也被认为具有相似的结构。一些金属离子，包括Lantanide离子，以类似于Ca~(2+)~(2+)和Gt~(2+)离子的方式与PLA2及其真正底物络合物结合，并产生PLA2活性(低于25%)。较少

1. Kinetics of the hydrolysis of monodispersed and micellar sustrates, catalyzed by Group I or II phospholipase A_2 (PLA_2s) in the presence of a saturating Ca^<2+> concentration indicated that deprotonated state of the catalytic group His 48 and protonated state of Tyr 52, the site of which is located in close proximity to the imidazole ring of His 48 were found to be essential for the catalysis. The importance of an ionized state of the N-terminal alpha-amino group at the active site was also indicated for Group I enzymes. The pK values of both His 48 and Tyr 52 of the enzyme-Ca^<2+> complex shifted markedly to the alkaline side on binding to micellar substrates, whereas no significant pK shifts were noted for the bindings to monodispersed substrates.2. Kinetic studies showed that Ca^<2+> binding to the both types of PLA_2s was essential for the catalysis. Substrate bindings to Group I PLA_2s were independent of the Ca^<2+> binding, whereas those for Group II enzymes were facilitated … More more than 10 times by the Ca^<2+> binging. The latter result was compatible with the hypothesis that an intermediate complex would be stabilized by coordination of the bound Ca^<2+> ion with the phosphoryl group and the carbonyl group at the sn-2 position of the substrate molecule. However, the former, the former result for Group I enzymes seemed incompatible for this mechanism. The X-ray crystallographic studies on the bindings of a substrate analog having an amide-bond instead of the sn-2 ester bond, dodecanoyl-2-aminohexanol-1-phosphoglycol, indicated that the carbonyl oxygen and phosphoryl moiety interact directly with the bound Ca^<2+> ion at the substrate binding site, suggesting that the binding of this analog should depend on the Ca^<2+> binding. Our studies in solution to confirm this showed the binding constants of this analog to both types of enzymes were increased significantly as the degrees of Ca^<2+> bindings increase, indicating that the structures of enzyme-substrate analog complex and enzyme-Ca^<2+>-analog complex in solution are very similar to each other as those in crystal and that the structures of the enzyme-analog complexes are stabilized by the Ca^<2+> binding. The enzyme-Ca^<2+>-genuine substrate complexes for the both types of PLA_2s are also considered to have similar structures.3. Some metal ions including lantanide ions were showed to bind to PLA_2s and to their genuine-substrate complexes in a manner similar to that of Ca^<2+> ion and to produce the PLA_2 activites (less than 25%). Less