半胱氨酸脱硫酶突变体H104Q,E156Q,D180G,Q183E和K206A通过改变对磷酸吡哆醛的结合影响酶活性

Altered Activities of Cysteine Desulfurase Variants H104Q,E156Q,D180G,Q183E and K206A Caused by Changes in Pyridoxal Phosphate Binding

大肠杆菌半胱氨酸脱硫酶(cysteine desulfurase,Isc S)是一类依赖磷酸吡哆醛(pyridoxal phosphate,PLP)的同质二聚体的酶.IscS能催化游离底物L-半胱氨酸脱硫,生成L-丙氨酸和单质硫.在此催化过程中,可形成与酶结合的半胱氨酸过硫化物中间物,并出现了7种具有不同特征性吸收峰的中间反应物.为了研究PLP的结合及中间反应物的形成及累积,对IscS中与PLP结合相关,及IscS半胱氨酸活性口袋中特定氨基酸残基位点(His104,Glu156,Asp180,Gln183和Lys206)进行定点突变,结果发现:1)IscS突变体H104Q、D180G、Q183E、K206A对PLP的结合能力具有不同程度的减弱,酶的活性明显降低甚至消失,PLP与蛋白结合的特异吸收峰消失,或发生明显偏移并出现新的吸收峰,且这些新出现的吸收峰又与蛋白形成的各种中间反应物的吸收峰一致;2)IscS突变体E156Q的活性增高,PLP与蛋白结合的吸收峰明显增加.这些结果都表明,IscS氨基酸残基可通过影响PLP的结合及质子转移引起催化过程中不同中间反应物的形成及累积,同时提高或降低蛋白的活性.

Cysteine desulfurase（ IscS）,a cysteine desulfurase in Escherichia coli,is pyridoxal 5＇-phosphate（ PLP）-dependent homodimeric enzyme. IscS catalyzes the L-cysteine conversion into L-alanine and sulfur. The formation of a protein-bound cysteine persulfide intermediate generates seven reactants with different absorption peaks. To study the PLP binding,intermediate formation and accumulation,we performed site-specific mutagenesison selected amino acid residues（ His104,Glu156,Asp180,Gln183 and Lys206）. In IscS cysteine active pocket,H104 Q,D180G,Q183 E,K206A reduced the binding abilities to PLP,and IscS activity significantly reduced as shown by the PLP absorption peaks. The E156 QIscS variant with the higher activitywas identified. The results showed that IscS amino acidresidues at the substrate binding site for proton transfer influenced the formation and accumulation of reaction intermediates.