Active site of bee venom phospholipase A(2): The role of histidine-34, aspartate-64 and tyrosine-87
Active site of bee venom phospholipase A(2): The role of histidine-34, aspartate-64 and tyrosine-87
复制标题
DOI：
10.1021/bi9528412
复制
发表时间：
1996-04-09
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：
Annand, RR;Kontoyianni, M;Gelb, MH
在蜂毒磷脂酶A（2）中，组氨酸-34可能作为一种布朗斯台德碱使攻击性水去质子化。天冬氨酸-64和酪氨酸-87与组氨酸-34形成氢键网络。我们在这些位置制备了突变体，并研究了它们的动力学性质。其中组氨酸-34变成谷氨酰胺的突变体没有催化活性，而天冬氨酸-64变成天冬酰胺或丙氨酸（界面转换数减少50-100倍）或酪氨酸-87变成苯丙氨酸（转换数没有变化）的突变体保留了良好的活性。所有突变体的界面米氏常数的变化小于10倍。分子模拟表明，天冬氨酸-64和酪氨酸-87的突变应该产生保留天然样结构和支持催化的酶。组氨酸-34咪唑的pK（a）由pH速率曲线和组氨酸-34烷基化速率对2-溴-4 '-硝基苯乙酮的pH依赖性推导得出。酪氨酸-87突变使pK（a）增加约半个单位，天冬氨酸-64突变为天冬酰胺使pK（a）减少约半个单位，而天冬氨酸-64突变为丙氨酸时pK（a）不变。这些pK（a）值与静电计算结果基本一致，表明天冬氨酸-64和组氨酸-34之间的氢键并不异常强。连接酪氨酸-87与天冬氨酸-64和天冬氨酸-64与组氨酸-34的氢键网络对于催化并不关键。
In bee venom phospholipase A(2), histidine-34 probably functions as a Bronsted base to deprotonate the attacking water. Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34. We have prepared mutants at these positions and studied their kinetic properties. The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis. The pK(a) of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone. The pK(a) is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pK(a) is unchanged. These pK(a)s are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong. The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.