The allosteric regulation of enzymeactivity is fundamental to preventing metabolic and homeostatic chaos. Acolossal number of complex, multifunctional enzymes important to metabolic andcellular processes exhibit some form of dynamic regulation, be it through small-moleculeeffector binding, protein-protein interactions or the multi-enzyme complexformation. The definition of "allostery" and "allosteric regulation" continuesto evolve with the advent of sophisticated biophysical techniques that candetermine the structural architecture of multifunctional proteins/proteincomplexes at increasing higher resolution and relate protein conformationalchanges and transient protein interactions to the regulation of catalyticactivity. The long-standing question of "How does protein structure dictate itsfunction?" has expanded to "How do protein structure, thermodynamics andconformational changes dictate the regulation of enzyme activity?"
While enzyme regulation key to maintaininghomeostasis, aberrant, or dysregulated enzyme activity is often the fundamentalbasis for the pathogenesis of numerous metabolic and neurodegenerativediseases. Our group is interested in untangling the complex mechanism ofaberrant enzyme activity, specifically in the context of metabolic disordersand neurodegenerative diseases. A complete understanding of the activity of theenzymes responsible for the pathogenesis of these diseases is critical toadvancing our fundamental understanding of disease progression and crucial tothe development of effective therapeutic modulators. Our current researchefforts are focused on determining the mechanisms governing the activity ofthree different enzyme/multi-enzyme complexes, each with progressively complicatedmodes of regulation.
Coordination of catalysis in Pyruvate Carboxylase (PC). Positionedat the crossroads of central metabolism, the activity of PC contributes to thepersistent hyperglycemia characteristic of type 2 diabetes. In hepatocytes, theprogression of type 2 diabetes results in marked increases in hepatic acetylCoenzyme A levels (acetyl-CoA) which results in the pathological activation ofPC and sustained increases in glucose production. In addition, PC is essential toglutamine-independent metabolism in cancerous tumor cells. The anpleroticactivity of PC compensates for decreased glutamine levels, allowing forincreased cell growth, proliferation and invasion. Tumor cells with high PC activityare often resistant to commonly used chemotherapeutics. While the underlyingmetabolic function of PC differs in hepatocytes and cancerous cells (i.e.gluconeogenesis vs. anaplerosis), the pathogenesis of both diseases is dependent on the regulation of PC activity.
There is little known about theoverall mechanism(s) regulating the activity of PC or other members of thebiotin-dependent carboxylase family. Previous studies from our lab and otherssuggest that the allosteric activator, acetyl-CoA, coordinates catalysisbetween the spatially distinct active sites in PC, linking the ATP-dependentcarboxylation of biotin in the biotin carboxylase (BC) domain with oxaloacetateproduction in the carboxyl transferase (CT) domain through the biotin carboxylcarrier (BCCP) domain. While acetyl-CoA is a powerful activator of the enzyme,our preliminary data also suggests that the regulation of activity is dependenton subunit interactions, thermodynamics, long-range active site communicationand substrate cooperativity, but the complex mechanism governing PC activity isunknown at the molecular level. Our preliminary data suggest that the activityof PC, as well as the family of biotin-dependent carboxylases, is regulated byinteractions between the functionally conserved BC and structurally conservedallosteric domain. In PC, these interactions are enhanced or stabilized in thepresence of acetyl-CoA.
Regulation of the MutuallyExclusive Activities of Transglutaminase 2 (TG2) by Calreticulin and Phospholipase Cδ 1. In the majority of late onset neurodegenerative diseases,the underlying cause of neuron degeneration is the accumulation of toxicprotein aggregates inside and outside of the neurons. The Ca2+-dependent,post-translational modification of tau, α-synuclein and β-amyloid bytransglutaminase 2 (TG2) contributes to the development of toxic proteinaggregates, plaques and tangles in the diseased brain. The physiological andpathological mechanisms of TG2 activity have been difficult to discern by invivo methods due, in part, to the multifunctionality of TG2. Not only doesTG2 possess Ca2+-dependent protein cross-linking activity, but italso exhibits GTPase activity that is important to cellular signaling. Thesemutually exclusive activities are reciprocally regulated such that GTP inhibitsthe Ca2+-dependent activity while Ca2+ inhibits GTPaseactivity. Conflicting invivo data has led to the current controversy surrounding thephysiological activity of TG2.
The imperative need forselectivity necessitates a working knowledge of both GTPase and Ca2+-dependentTG2 activity under physiological and pathological conditions and the ability todistinguish the relative effects of therapeutics on each activity. Our long-termresearch goal is to identify and define the molecularmechanisms responsible for the governance of physiological and pathological TG2activity, which we believe is to be regulated not only by GTP and Ca2+,but also through specific interactions with calreticulin and phospholipase Cδ1.
Determiningthe Kinetic Advantage to Mitochondrial Respiratory Supercomplex Formation. Cellular respiration in themitochondria of higher organisms is paramount to supplying the ATP needed tosustain biological processes. The redox reactions of the mitochondrialrespiratory chain, which consists of four transmembrane enzyme complexes, a lipid-solubleelectron carrier (ubiquinone), and a water-soluble electron carrier (cytochromec), generate the electrochemical proton gradient necessary to drive ATPsynthesis. While ample evidence has shown that these enzymes exist both assolitary units and supercomplex assemblies in the inner mitochondrial membrane,controversy surrounds the functional and kinetic advantage gained by theformation of the mutli-enzyme supercomplexes. To aid in resolving thecontroversy, we have an initiated an in-depth steady-state and pre-steady statekinetic analysis of the enzyme activity ofintact mitochondria to determine if there is any kinetic or regulatoryadvantage to complex formation. Our future goals will also use novelbiophysical techniques to probe the nature of the transient proteininteractions governing supercomplex formation in both physiological andpathological states.
Techniques. 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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Shaikh, S.R., Sullivan, E.M., Alleman, R.J., Brown, D.A., Zeczycki, T.N. (2014) Increasing mitochondrial membrane phospholipid content lowers the enzymatic activity of electron transport complexes. Biochemistry 53, 5589-5591.
Zeczycki, T.N., Whelan, J., Hayden, W.T., Brown, D.A., Shaikh, S.R. (2014) Increasing levels of cardiolipin differentially influences packing of phospholipids found in the mitochondrial inner membrane. Biochem. Biophys. Res. Com. 450, 366-371.
Marlier, J.F., Cleland, W.W., Zeczycki, T.N. (2013) Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli. Biochemistry 52, 2888-2894
Adina-Zada, A., Seeruk, C., Jitrapakdee, S., Zeczycki, T.N., St. Maurice, M., Cleland, W.W., Wallace, J.C., Attwood, P.V. (2012) Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase. Biochemistry 51, 8208-8217. PMCID PMC3567212.
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
Menefee, A.L., Zeczycki, T.N. (2014) Nearly 50 Years in the Making: Defining the Catalytic Mechanism of the Multifunctional Enzyme, Pyruvate Carboxylase. FEBS J 281, 1333-1354.
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.
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.
Assistant Professor of Biochemistry & Molecular Biology
ECDOI Office 4116 (Lab 4102-61) MS743
Biochemistry & Molecular Biology, BSOM at East Carolina University
Greenville, NC 27834