Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

详细信息    查看全文

• 作者：Tonya N. Zeczycki ; Ann L. Menefee ; Sarawut Jitrapakdee ; John C. Wallace ; Paul V. Attwood ; Martin St. Maurice ; W. Wallace Cleland
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：November 15, 2011
• 年：2011
• 卷：50
• 期：45
• 页码：9694-9707
• 全文大小：530K
• 年卷期：v.50,no.45(November 15, 2011)
• ISSN：1520-4995

文摘

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg²⁺, and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg²⁺ assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg²⁺ vs pyruvate were nonlinear at low concentrations of Mg²⁺ and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg²⁺ also resulted in a decrease in the K_a for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K_i = 0.026 mM) and pyruvate carboxylation (K_i = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg²⁺ metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg²⁺ are described.