Pyruvate carboxylase is a member of the family of biotin-dependent enzymes which play key roles in intermediary metabolism. Pyruvate carboxylase deficiencies mostly occur in neonates or infants and result in severe psychomotor retardation leading to early death. Over expression of hepatic pyruvate carboxylase is associated with type II diabetes and obesity. The purpose of this project is to determine the first three dimensional structure of pyruvate carboxylase as an example of an intact biotin-dependent holoenzyme, and to perform a detailed structure-function analysis on this enzyme. We shall: (1) Perform crystallization trials on pyruvate carboxylases from a number of sources with the aim of determining the 3-D structure of the enzyme by X-ray crystallographic analysis. (2) Determine the binding site of the physiological, allosteric activator, acetyl CoA by co-crystallization with a non-hydrolysable analogue and by affinity labeling. (3) Determine the regions of polypeptide sequence involved in the action of acetyl CoA by producing chimeric constructs of pyruvate carboxylases from species which show different degrees of response to acetyl CoA. By performing site-directed mutagenesis on specific amino acid residues in the enzyme and kinetic analyses of the wild-type and mutant enzymes using a combination of 2H, 13C and 18O kinetic isotope effect experiments, and steady-state and pre-steady state kinetic analysis to pinpoint the precise roles of these residues in the catalytic mechanism of the enzyme we shall elucidate (4) The catalytic mechanism of the biotin carboxylation reaction common to the majority of biotin-dependent carboxylases in the context of a holoenzyme. (5) The catalytic mechanism of the carboxylation of pyruvate by carboxybiotin and movement of carboxybiotin between the sites of the partial reactions in the enzyme active site. (6) The action of acetyl CoA at the level of the catalytic mechanism. (7) We shall synthesize carboxyphosphate and demonstrate its ability to act as an intermediate in the pyruvate carboxylase reaction.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BIO (01))
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Jones, Warren
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University of Wisconsin Madison
Other Domestic Higher Education
United States
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Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul et al. (2015) Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation. FEBS Lett 589:2073-9
Sirithanakorn, Chaiyos; Adina-Zada, Abdussalam; Wallace, John C et al. (2014) Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate. Biochemistry 53:7100-6
Lietzan, Adam D; Lin, Yi; St Maurice, Martin (2014) The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase. Arch Biochem Biophys 562:70-9
Adina-Zada, Abdussalam; Jitrapakdee, Sarawut; Wallace, John C et al. (2014) Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase. Biochemistry 53:1051-8
Marlier, John F; Cleland, W W; Zeczycki, Tonya N (2013) Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli. Biochemistry 52:2888-94
Lietzan, Adam D; St Maurice, Martin (2013) A substrate-induced biotin binding pocket in the carboxyltransferase domain of pyruvate carboxylase. J Biol Chem 288:19915-25
Lietzan, Adam D; St Maurice, Martin (2013) Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs. Biochem Biophys Res Commun 441:377-82
Adina-Zada, Abdussalam; Sereeruk, Chutima; Jitrapakdee, Sarawut et al. (2012) Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase. Biochemistry 51:8208-17
Sivadas, Priyanka; Dienes, Jennifer M; St Maurice, Martin et al. (2012) A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex. J Cell Biol 199:639-51
Adina-Zada, Abdussalam; Zeczycki, Tonya N; St Maurice, Martin et al. (2012) Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA. Biochem Soc Trans 40:567-72

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