Xu Wenxin, Ahmed Shaheen, Moriyama Hideaki, Chollet Raymond
Department of Biochemistry, Lincoln, Nebraska 68588-0664.
Department of Chemistry, Lincoln, Nebraska 68588-0664.
J Biol Chem. 2006 Jun 23;281(25):17238-17245. doi: 10.1074/jbc.M602299200. Epub 2006 Apr 18.
Phosphoenolpyruvate carboxylase (PEPC) is a "multifaceted," allosteric enzyme involved in C4 acid metabolism in green plants/microalgae and prokaryotes. Before the elucidation of the three-dimensional structures of maize C4 leaf and Escherichia coli PEPC, our truncation analysis of the sorghum C4 homologue revealed important roles for the enzyme's C-terminal alpha-helix and its appended QNTG961 tetrapeptide in polypeptide stability and overall catalysis, respectively. Collectively, these functional and structural observations implicate the importance of the PEPC C-terminal tetrapeptide for both catalysis and negative allosteric regulation. We have now more finely dissected this element of PEPC structure-function by modification of the absolutely conserved C-terminal glycine of the sorghum C4 isoform by site-specific mutagenesis (G961(A/V/D)) and truncation (DeltaC1/C4). Although the C4 polypeptide failed to accumulate in a PEPC- strain (XH11) of E. coli transformed with the Asp mutant, the other variants were produced at wild-type levels. Although neither of these four mutants displayed an apparent destabilization of the purified PEPC homotetramer, all were compromised catalytically in vivo and in vitro. Functional complementation of XH11 cells under selective growth conditions was restricted progressively by the Ala, DeltaC1 and Val, and DeltaC4 modifications. Likewise, steady-state kinetic analysis of the purified mutant enzymes revealed corresponding negative trends in kcat and kcat/K0.5 (phosphoenolpyruvate) but not in K0.5 or the Hill coefficient. Homology modeling of these sorghum C-terminal variants against the structure of the closely related maize C4 isoform predicted perturbations in active-site molecular cavities and/or ion-pairing with essential, invariant Arg-638. These collective observations reveal that even a modest, neutral alteration of the PEPC C-terminal hydrogen atom side chain is detrimental to enzyme function.
磷酸烯醇式丙酮酸羧化酶（PEPC）是一种“多功能”的别构酶，参与绿色植物/微藻和原核生物的C4酸代谢。在阐明玉米C4叶片和大肠杆菌PEPC的三维结构之前，我们对高粱C4同源物进行的截短分析表明，该酶的C端α-螺旋及其附加的QNTG961四肽分别在多肽稳定性和整体催化中发挥重要作用。总的来说，这些功能和结构观察结果表明PEPC C端四肽对催化和负别构调节都很重要。我们现在通过定点诱变（G961(A/V/D)）和截短（DeltaC1/C4）对高粱C4同工型绝对保守的C端甘氨酸进行修饰，更精细地剖析了PEPC结构-功能的这一元件。尽管用天冬氨酸突变体转化的大肠杆菌PEPC菌株（XH11）中C4多肽未能积累，但其他变体以野生型水平产生。尽管这四个突变体均未显示纯化的PEPC同四聚体有明显的不稳定，但在体内和体外其催化作用均受到损害。在选择性生长条件下，XH11细胞的功能互补因丙氨酸、DeltaC1和缬氨酸以及DeltaC4修饰而逐渐受到限制。同样，对纯化的突变酶进行的稳态动力学分析显示，kcat和kcat/K0.5（磷酸烯醇式丙酮酸）呈相应的负趋势，但K0.5或希尔系数无此趋势。将这些高粱C端变体与密切相关的玉米C4同工型结构进行同源建模，预测活性位点分子腔和/或与必需的不变精氨酸-638的离子配对会受到干扰。这些综合观察结果表明，即使PEPC C端氢原子侧链发生适度的中性改变也会损害酶的功能。
