Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase

• Published in: The FEBS journal Vol. 281; no. 5; pp. 1333 - 1354
• Main Authors: Menefee, Ann L., Zeczycki, Tonya N.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.03.2014
• Subjects: allostery; Amino Acid Sequence; Animals; Carboxylation; Catalysis; Catalytic Domain; coordination of catalysis; Coupling (molecular); Enzymes; Holoenzymes - chemistry; Holoenzymes - genetics; Holoenzymes - metabolism; Humans; Kinetics; Models, Molecular; Molecular biology; Molecular modelling; Molecular Sequence Data; multifunctional enzymes; Multifunctional Enzymes - chemistry; Multifunctional Enzymes - genetics; Multifunctional Enzymes - metabolism; nonclassical ping‐pong mechanism; nonlinear initial velocity plots; Product inhibition; Protein Structure, Tertiary; Pyruvate carboxylase; Pyruvate Carboxylase - chemistry; Pyruvate Carboxylase - genetics; Pyruvate Carboxylase - metabolism; Pyruvic acid; Reaction kinetics; Reaction mechanisms; Sequence Homology, Amino Acid; sequential mechanism; steady‐state kinetics; Substrate inhibition; nonclassical ping-pong mechanism; coordination of catalysis; sequential mechanism; pyruvate carboxylase; nonlinear initial velocity plots; steady-state kinetics; allostery; multifunctional enzymes
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12713

Numerous steady‐state kinetic studies have examined the complex catalytic reaction mechanism of the multifunctional enzyme, pyruvate carboxylase (PC). Through initial velocity, product inhibition, isotopic exchange and alternate substrate experiments, early investigators established that PC catalyzes the MgATP‐dependent carboxylation of pyruvate by HCO3– through a nonclassical sequential Bi Bi Uni Uni reaction mechanism. This review surveys previous steady‐state kinetic investigations of PC and evaluates the proposed hypotheses concerning the overall catalytic mechanism, nonlinear kinetics and active site coupling in the context of recent structural and mutagenic analyses of this multifunctional enzyme. The determination several PC holoenzyme structures have aided in corroborating the proposed molecular mechanisms by which catalysis occurs and established the inextricable link between the dynamic protein motions and complex kinetic mechanisms associated with PC activity. Unexpectedly, the conclusions drawn from these early steady‐state kinetic investigations have consistently proven to be in fundamental agreement with our current understanding of PC catalysis, which is a testament to the overarching sophistication of the methods pioneered by Michaelis and Menten and further developed by Northrop, Cleland and others. Steady‐state kinetic studies have examined the complex catalytic mechanism of the pyruvate carboxylase (PC). Several PC holoenzyme structures have aided in corroborating the proposed mechanisms. Surprisingly, these early investigations have consistently proven to be in agreement with our current understanding of PC, a testament to the overarching sophistication of the methods pioneered by Michaelis and Menten, Northrop, Cleland and others.