Evidence for a functional role for histidine in lysyl oxidase catalysis.
Evidence for a functional role for histidine in lysyl oxidase catalysis.
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DOI：
10.1016/s0021-9258(18)37447-7
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发表时间：
1988-11
期刊：
影响因子：
--
通讯作者：
S. Gacheru;P. Trackman;H. Kagan
中科院分区：
文献类型：
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作者：
S. Gacheru;P. Trackman;H. Kagan
考察了赖氨酰氧化酶催化的pH依赖动力学，以寻找可电离的酶残基的证据，该酶残基可能作为一般碱催化质子提取，这是该酶处理底物机制的一个组成部分。正己胺氧化的对数Vmax/Km与pH的关系曲线图得到pKa值分别为7.0+/-0.1和10.4+/-0.1。不同底物的pKa值不同，反映了底物氨基的电离。较低pKa随温度变化的van‘t Hoff图给出的电离热为6.1kcal·mol-1。这一值以及pKa为7.0，与以前被认为是酶中通用碱基催化剂的组氨酸残基的pKa一致。赖氨酰氧化酶与低浓度的组氨酸选择性试剂焦碳酸二乙酯在22℃和pH 7.0下孵育时，以假一级动力学过程不可逆地抑制酶的活性。赖氨酰氧化酶的失活与1组氨酸残基/摩尔酶化学修饰的光谱和依赖于pH的动力学证据相关联，其pKa为6.9+/-0.1，其实验误差与对数Vmax/Km随pH变化的曲线图中的结果一致。经修饰的酶与羟胺孵育可恢复酶活性，这与该亲核试剂取代N-甲氧基组氨酸中的甲氧基的能力一致。正己胺底物的存在在很大程度上防止了焦碳酸二乙酯对酶的失活。因此，这些结果表明组氨酸在赖氨酰氧化酶催化中的功能作用与一般碱在质子提取中的作用一致。
The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.