Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase.
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.
Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.|The East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University, Greenville, NC, USA.
Ann L
AL
Menefee
Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA.|The East Carolina Diabetes and Obesity Institute, Brody School of Medicine at East Carolina University, Greenville, NC, USA.
Tonya N
TN
Zeczycki
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D064251:Multifunctional Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D020035:Holoenzymes; D064251:Multifunctional Enzymes; D011766:Pyruvate Carboxylase
allostery; coordination of catalysis; multifunctional enzymes; nonclassical ping-pong mechanism; nonlinear initial velocity plots; pyruvate carboxylase; sequential mechanism; steady-state kinetics