组氨酸残基在焦磷酸:果糖-6-磷酸1-磷酸转移酶催化功能中的作用

应用焦碳酸二乙酯(DEPC)对焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP)进行化学修饰时,酶的活性迅速丧失,呈拟一级动力学反应.羟胺的复活作用及修饰后酶的紫外光谱变化表明,酶失活的原因是DEPC与酶的组氨酸残基形成了乙酯基组氨酸复合物.失活动力学表明,酶的活性位点结合1分子DEPC即丧失活性.活性位点的组氨酸残基的完整性是酶活性的必要条件之一.底物F6P、产物果糖1,6-二磷酸(FBP)、P i及激活剂果糖2,6-二磷酸(F2,6BP)均可保护酶免被DEPC失活,底物PP i却没有保护作用.组氨酸残基在酶的催化功能中可能与F6P,FBP及P i的结合过程有关,而与PP i无关.

Role of histidine residues in the catalytic activity of PFP from pineapple leaves

Diethylpyrocarbonate(DEPC) inactivated pineapple leaf pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase(PFP,EC 2.7.1.90). The inactivation kinetics of the enzyme by DEPC was pseudo-first order one with respect to incubation time and inhibitor concentration.Activity of modified PFP could be restored by treatment with hydroxylamine.The ultraviolet difference spectrum of modified(versus) native enzyme revealed the formation of ethoxyformal histidine residues during inactivation.Kinetic data of figure 2 indicates that only one molecule of DEPC is involved in binding to each active unit of enzyme during inactivation.The substrate fructose-6-phosphate(F6P),the products fructose-1,6-bisphosphate(FBP) and phosphate(Pi) protected PFP against the DEPC modification,suggesting that the modified histidine residue resides at active sites.Another substrate pyrophosphate(PPi) had no protective effect on the exzyme,indicating that the histidine residue at the active site is not involved in the binding with PPi.