Essential histidine residues of ribulosebisphosphate carboxylase indicated by reaction with diethylpyrocarbonate and rose bengal

A.S. Bhagwat, J. Ramakrishna
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引用次数: 15

Abstract

Modification of ribulosebisphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) by diethylpyrocarbonate or rose bengal-sensitized photooxidation caused rapid inactivation of the enzyme. The photooxidation proceeded following pseudo-first-order reaction kinetics showing a maximal value at pH 8.0. The fully activated enzyme was more sensitive to photooxidation as compared to the unactivated enzyme. The enzyme partially inactivated by photooxidation was fully sensitive to the positive effectors. The photooxidised enzyme showed a characteristic increase in absorbance at 250 nm which was dependent on the extent of inactivation. The kinetic analyses and correlation of the spectral changes with the activity indicated that the inactivation by diethylpyrocarbonate resulted from the modification of an average one histidine residue/70 000-dalton combination of large and small subunit. Sulfhydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. Ribulosebisphosphate and some effectors of the enzyme offered significant protection against diethylpyrocarbonate modification indicating that diethylpyrocarbonate was interacting with the enzyme at or near the active site.

核酮糖二磷酸羧化酶必需组氨酸残基与焦碳酸二乙酯和孟加拉玫瑰的反应
二磷酸核酮糖羧化酶(3-磷酸-d-甘油羧化酶(二聚体),EC 4.1.1.39)被焦碳酸二乙酯或玫瑰红敏化光氧化修饰导致酶快速失活。光氧化过程遵循准一级反应动力学,在pH 8.0时达到最大值。与未激活的酶相比,完全激活的酶对光氧化更敏感。被光氧化部分失活的酶对正效应物完全敏感。光氧化酶在250 nm处表现出特征的吸光度增加,这取决于失活程度。动力学分析和光谱变化与活性的相关性表明,焦碳酸二乙酯的失活是由于修饰了平均1个组氨酸残基/7万道尔顿的大小亚基组合。巯基、赖氨酸和酪氨酸残基未被焦碳酸二乙酯处理过。二磷酸核酮糖和酶的一些效应物对焦碳酸二乙酯修饰具有显著的保护作用,表明焦碳酸二乙酯在活性位点或活性位点附近与酶相互作用。
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