精氨酸残基和赖氨酸残基对大鼠血红素加氧酶-1催化功能的影响

目的研究和探讨大鼠血红素加氧酶-1(RHO-1)中保守的碱性氨基酸精氨酸残基和赖氨酸残基对该酶催化功能的影响。方法应用定点诱变制备20种RHO-1的变异型酶质粒，其中RHO-1中保守的碱性氨基酸精氨酸和赖氨酸被置换成谷氨酸，将它们在大肠埃希菌中表达并纯化后，用光谱扫描方法在以NADPH-细胞色素P-450还原酶提供电子的模拟体内反应体系下测定变异型酶和野生型酶的活性，从而观察变异型酶的活性是否发生改变。结果变异型酶R35E、K39E、R44E、R136E、K148E、K149E、K153E、R185E和K196E对血红素的降解速度低于野生型酶。结论R35等9个精氨酸残基和赖氨酸残基在参与RHO-1催化血红素降解的反应中对该酶的催化功能有着重要影响。

Effect of arginine and lysine residues on catalytic function of rat heme oxygenase-1

Objective To study the catalytic function of conservative basic amino acid arginine residue and lysine residue on rat heme oxygenase-1(RHO-1). Methods We prepared 20 mutants of RHO-1 in which conservative basic amino acids Arg and Lys were changed to Glu by site-mutagenesis. They were expressed in Escherichia coli, purified and estimated catalytic activity by spectrum scan assay with simulating body reaction system. Results The levels of hemin reduction for mutants R35E, K39E, R44E, R136E, K148E, K149E, K153E, R185E and K196E were lower compared to wild RHO-1. Conclusions R35, K39, R44, R136, K148, K149, K153, R185 and K196 have important effect on catalytic function in the reaction of hemin reduction.