禽肝磷酸烯醇丙酮酸羧激酶中的一个活性位点赖氨酸。

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase.

作者信息

Guidinger P F, Nowak T

机构信息

Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.

出版信息

Biochemistry. 1991 Sep 10;30(36):8851-61. doi: 10.1021/bi00100a018.

DOI:10.1021/bi00100a018

PMID:1909575

Abstract

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

摘要

通过化学修饰和对修饰酶的特性分析，研究了赖氨酸在禽肝磷酸烯醇丙酮酸羧激酶催化过程中的作用。2,4-戊二酮使酶失活的速率为假一级反应，且与试剂浓度呈线性相关，二级反应速率常数为0.36±0.025 M⁻¹ min⁻¹。磷酸烯醇丙酮酸羧激酶催化的可逆反应被磷酸吡哆醛5'-磷酸失活遵循双分子动力学，二级反应速率常数为7700±860 M⁻¹ min⁻¹。该酶催化的不可逆反应的失活二级反应速率常数为1434±110 M⁻¹ min⁻¹。用磷酸吡哆醛5'-磷酸处理该酶，每摩尔酶掺入1摩尔磷酸吡哆醛5'-磷酸，即一个赖氨酸残基被修饰，同时活性丧失100%。当监测该酶催化的可逆或不可逆反应时，观察到化学计量比为1:1。对观测速率常数与pH的研究表明，这个活性位点赖氨酸的pKa为8.1，与pH无关的失活速率常数为47700 M⁻¹ min⁻¹。含磷酸的底物IDP、ITP和磷酸烯醇丙酮酸对磷酸吡哆醛5'-磷酸引起的失活几乎提供完全保护。如电子顺磁共振所示，修饰后的无活性酶在锰离子结合方面几乎没有变化。质子弛豫速率测量表明，磷酸吡哆醛5'-磷酸修饰改变了含磷酸底物的结合。³¹P NMR弛豫速率测量显示，在三元酶-Mn²⁺-底物复合物中底物的结合发生了改变。圆二色性研究表明，磷酸吡哆醛5'-磷酸修饰的磷酸烯醇丙酮酸羧激酶的二级结构几乎没有变化。这些结果表明，禽肝磷酸烯醇丙酮酸羧激酶在活性位点有一个反应性赖氨酸，它参与含磷酸底物的结合和活化。