Fukuda H, Sopena de Kracoff Y E, Iñigo L E, Paredes S R, Ferramola de Sancovich A M, Sancovich H A, Batlle A M
Department of Biochemistry, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Buenos Aires, Argentine.
J Enzyme Inhib. 1990;3(4):295-302. doi: 10.3109/14756369009030378.
Photoxidation with methylene blue and rose bengal and chemical modification by diethylpryrocarbonate of pig liver 5-aminolevulinic acid dehydratase produced strong inactivation of the enzyme which was concentration dependent. Loss of enzyme activity by both photoxidation and ethoxyformylation was pH and time-dependent and protected by the presence of the substate and competitive inhibitors. The rate of inactivation was directly related to the state of protonation of histidyl groups, the unprotonated from being modified at a much faster rate than the protonated form. Plots of the pseudo-first order rate constants for 5-aminolevulinic acid dehydratase inactivation against pH resulted in typical titration curves showing inflection points at about pH 6.4 for methylene blue and rose bengal and 6.8 for diethylprocarbonate providing further and unequivocal evidence for the existence of critical histidyl groups at the active centre of the enzyme.
用亚甲蓝和孟加拉玫瑰红进行光氧化以及用焦碳酸二乙酯对猪肝5-氨基乙酰丙酸脱水酶进行化学修饰，会使该酶产生强烈失活，且这种失活具有浓度依赖性。光氧化和乙氧基甲酰化导致的酶活性丧失与pH值和时间有关，并受到底物和竞争性抑制剂的保护。失活速率与组氨酸基团的质子化状态直接相关，未质子化形式的修饰速率比质子化形式快得多。以5-氨基乙酰丙酸脱水酶失活的伪一级速率常数对pH值作图，得到典型的滴定曲线，亚甲蓝和孟加拉玫瑰红的拐点约为pH 6.4，焦碳酸二乙酯的拐点为pH 6.8，这为该酶活性中心存在关键组氨酸基团提供了进一步的确凿证据。
