Characterizing the Molecular Basis of the Allosteric Activation of Pyruvate Carboxylase by Acetyl CoA

Abstract

Pyruvate carboxylase (PC) is a central metabolic enzyme that produces oxaloacetate from pyruvate and bicarbonate in an ATP-dependent manner. The catalytic activity of PC contributes to glucose homeostasis and has been implicated in enhanced tumorigenesis and metastasis in certain cancers. PC activity is allosterically activated by acetyl CoA, and allosterically inhibited by L-aspartate. The binding of these effector molecules is mutually exclusive. The binding site for acetyl-CoA has largely been identified through previous structural studies, but the exact binding site for the acetyl moiety is unknown, partly as a consequence of the enzyme-catalyzed hydrolysis of acetyl CoA. The acetyl moiety is a particularly important feature of acetyl CoA activation, contributing to the significantly enhanced allosteric activation of acetyl CoA relative to CoA. Here, we have employed non-hydrolysable and truncated analogs of acetyl-CoA to more precisely probe the acetyl moiety binding site in PC. We have determined that the acetyl moiety contributes greatly to the binding of the molecule but that it does not alter the degree of activation. Additionally, kinetic studies strongly suggest that L-aspartate competes for the acetyl moiety binding site with acetyl CoA. A definition of the acetyl moiety binding site further clarifies the mechanism of acetyl CoA binding and offers a more detailed molecular description of allosteric regulation in this central metabolic enzyme.