The biochemical basis for the pathogenesis of Type 2 diabetes and the metabolic syndrome is a function of aberrant enzymatic activi-ties associated with glucose and lipid metabolism and free fatty acid synthesis/degradation. We are particularly interested in determin-ing the mechanisms of catalysis and unraveling the complexities of allosteric regulation of those multifunctional enzymes important to metabolic processes. A complete understanding of the activity and regulation of these enzymes is critical to the development of effec-tive therapeutic modulators for the treatment of metabolic disorders. The current focus of research in our lab is directed towards the characterization of the biochemical transformations and coordination of catalysis in several different multifunctional enzymes includ-ing pyruvate carboxylase (PC), peroxisomal multifunctional enzymes 1 and 2 (pMFE1 and pMFE2) and mitochondrial trifrunctional protein (mMTP).
PC, a regulator of gluconeogenesis in the liver and an anplerotic enzyme in the pancreatic β-cells, catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate. Abnormal PC activity is correlated directly with a reduction in glucose-stimulated insulin secretion and an increase in hepatic gluconeogenesis. In conjunction with our collaborators, we have used site-directed mutagenesis, steady-state kinetic studies and crystallography to establish the kinetic and chemical reaction that occurs in each of the active sites. Our focus has now shifted to resolving the mechanism of communication, coordination of catalysis and regulation of the activities occurring in the two spatially distinct active sites.
Alterations in peroxisomal and mitochondrial multifunctional enzymatic activities that are responsible for catalyzing key steps in the fatty acid β-oxidation pathway contribute to increased lipotoxicity in the liver and stimulation of glucose synthesis. β-oxidation of fatty acids in the mitochondria generates acetyl-CoA, the allosteric activator of PC, while β-oxidation in the peroxisome generates acetate. pMFE1 is a monomeric protein that shows catalytic activities analogous to the homologous monofunctional ∆3, ∆2-enoyl CoA isomerase, 2-enoyl CoA hydratase (crotonase) and (3S)-hydroxyacyl CoA dehydrogenase enzymes. The simultaneous existence of pMFE1 and pMFE2 is intriguing considering that both enzymes catalyze the hydration of 2-enoyl-CoA and dehydrogenation of the hydroxyl-acyl CoA product but exhibit a complete lack of structural homology. In the mitochondria, the α4β4octameric mMTP pro-tein exhibits 2-enoyl hydratase and NAD+-dependent (3S)-hydroxyl acyl dehydrogenase activity in the α-subunit. The β-subunit ex-hibits CoA-dependent 3-ketothiolase activity. The role of these enzymes in lipid metabolism makes them novel and attractive targets for the development of therapeutic modulators for the treatment of Type 2 diabetes and the metabolic syndrome.
In order to determine the physiologically relevant kinetic, chemical and allosteric mechanisms inherent to these enzymes, our lab uses a combination of in vitrotechniques to examine the unique enzymatic biochemical transformations, dynamic domain motions and inter-subunit communication pathways. These techniques, common to the fields of molecular biology, chemical biology and enzymol-ogy, include protein engineering, site-directed mutagenesis, steady-state and pre-steady-state kinetics, FRET, and 1D/2D NMR.
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Adina-Zada, A., Zeczycki, T.N., St. Maurice, M., Jitrapakdee, S., Cleland, W. W., Attwood, P.V. (2012) Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA. Biochem. Soc. Trans. 40, 567-572.
Zeczycki, T. N., Menefee, A.L, Jitrapakdee, S., Wallace, J.C., Attwood, P.V., St. Maurice, M., Cleland, W.W. (2011) Activation and inhibition of pyruvate carboxylase from Rhizobium etli. Biochemistry, 50, 9694-96707; PMID: 21958066
Zeczycki, T.N., Menefee, A. L., Adina-Zada, A., Jitrapakdee, S., Surinya, K.H., Wallace, J.C., Attwood, P.V., St. Maurice, M., Cle-land, W. W. (2011) Novel insights into the biotin carboxylase reaction of pyruvate carboxylase from Rhizobium etli.Biochemistry, 50, 9724-9737. PMID: 21957995
Lietzen, A., Menefee, A. L., Zeczycki, T. N., Kumar, S., Jitrapakdee, S., Attwood, P. V., Wallace, J. C., Cleland, W. W., St. Maurice, M. (2011) Interaction between the biotin carrier domain and the biotin carboxylase domain in the structure ofRhizobium etli Pyruvate Carboxylase. Biochemistry, 50, 9708-9723. PMID: 21958016
Adina-Zada, A., Hazra, R., Sereeruk, C., Jitrapakdee, S., Zeczycki, T. N., St. Maurice, M., Cleland, W.W., Wallace, J. C., Attwood, P.V. (2011) Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA. Arch. Biochem. Biophys. 509, 117-126. PMID:21426897
Duangpan, S., Jitrapakdee, S, Adina-Zada, A., Byrne, L., Zeczycki, T. N., St. Maurice, M., Cleland, W. W., Wallace, J. C., Attwood, P. V. (2010) Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of theRhizobium etli pyruvate car-boxylase by site-directed mutagenesis. Biochemistry, 49, 3296-3304. PMID: 20230056
Zeczycki, T. N., St. Maurice, M., Jitrapakdee, S., Wallace, J.C., Attwood, P.V., Cleland, W.W. (2009) Insight into the carboxyl trans-ferase domain mechanism of pyruvate carboxylase from Rhizobium etli. Biochemistry, 48, 4305-4313. PMID: 19341298
Adina-Zada, A., Zeczycki, T.N., Attwood, P.V. (2011) Regulation of the structure and activity of pyruvate carboxylase by acetyl-CoA. Arch. Biochem. Biophys. 519, 118-130. PMID: 22120519.
Zeczycki, T. N., St. Maurice, M., Attwood, P.V. (2010) Inhibitors of pyruvate carboxylase. Open Enzyme Inhib. J., 3, 8-26. PMID: 22180764
Assistant Professor of Biochemistry & Molecular Biology
ECDOI Office 4116 (Lab 4102-61) MS743
Biochemistry & Molecular Biology, BSOM at East Carolina University
Greenville, NC 27834