The allosteric regulation of enzyme activity is fundamental to preventing metabolic and homeostatic chaos. A colossal number of complex, multifunctional enzymes important to metabolic and cellular processes exhibit some form of dynamic regulation, be it through small-molecule effector binding, protein-protein interactions or the multi-enzyme complex formation. The definition of "allostery" and "allosteric regulation" continues to evolve with the advent of sophisticated biophysical techniques that can determine the structural architecture of multifunctional proteins/protein complexes at increasing higher resolution and relate protein conformational changes and transient protein interactions to the regulation of catalytic activity. The long-standing question of "How does protein structure dictate its function?" has expanded to "How do protein structure, thermodynamics and conformational changes dictate the regulation of enzyme activity?"
While enzyme regulation key to maintaining homeostasis, aberrant, or dysregulated enzyme activity is often the fundamental basis for the pathogenesis of numerous metabolic and neurodegenerative diseases. Our group is interested in untangling the complex mechanism of aberrant enzyme activity, specifically in the context of metabolic disorders and neurodegenerative diseases. A complete understanding of the activity of the enzymes responsible for the pathogenesis of these diseases is critical to advancing our fundamental understanding of disease progression and crucial to the development of effective therapeutic modulators. Our current research efforts are focused on determining the mechanisms governing the activity of three different enzyme/multi-enzyme complexes, each with progressively complicated modes of regulation.
Coordination of catalysis in Pyruvate Carboxylase (PC). Positioned at the crossroads of central metabolism, the activity of PC contributes to the persistent hyperglycemia characteristic of type 2 diabetes. In hepatocytes, the progression of type 2 diabetes results in marked increases in hepatic acetyl Coenzyme A levels (acetyl-CoA) which results in the pathological activation of PC and sustained increases in glucose production. In addition, PC is essential to glutamine-independent metabolism in cancerous tumor cells. The anplerotic activity of PC compensates for decreased glutamine levels, allowing for increased cell growth, proliferation and invasion. Tumor cells with high PC activity are often resistant to commonly used chemotherapeutics. While the underlying metabolic 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 the overall mechanism(s) regulating the activity of PC or other members of the biotin-dependent carboxylase family. Previous studies from our lab and others suggest that the allosteric activator, acetyl-CoA, coordinates catalysis between the spatially distinct active sites in PC, linking the ATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with oxaloacetate production in the carboxyl transferase (CT) domain through the biotin carboxyl carrier (BCCP) domain. While acetyl-CoA is a powerful activator of the enzyme,our preliminary data also suggests that the regulation of activity is dependent on subunit interactions, thermodynamics, long-range active site communication and substrate cooperativity, but the complex mechanism governing PC activity is unknown at the molecular level. Our preliminary data suggest that the activity of PC, as well as the family of biotin-dependent carboxylases, is regulated by interactions between the functionally conserved BC and structurally conserved allosteric domain. In PC, these interactions are enhanced or stabilized in the presence of acetyl-CoA.
Regulation of the Mutually Exclusive 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 toxic protein aggregates inside and outside of the neurons. The Ca2+-dependent, post-translational modification of tau, α-synuclein and β-amyloid by transglutaminase 2 (TG2) contributes to the development of toxic protein aggregates, plaques and tangles in the diseased brain. The physiological and pathological mechanisms of TG2 activity have been difficult to discern by in vivo methods due, in part, to the multifunctionality of TG2. Not only doesTG2 possess Ca2+-dependent protein cross-linking activity, but it also exhibits GTPase activity that is important to cellular signaling. These mutually exclusive activities are reciprocally regulated such that GTP inhibits the Ca2+-dependent activity while Ca2+ inhibits GTPase activity. Conflicting in vivo data has led to the current controversy surrounding the physiological activity of TG2.
The imperative need for selectivity necessitates a working knowledge of both GTPase and Ca2+-dependent TG2 activity under physiological and pathological conditions and the ability to distinguish the relative effects of therapeutics on each activity. Our long-term research goal is to identify and define the molecular mechanisms responsible for the governance of physiological and pathological TG2 activity, which we believe is to be regulated not only by GTP and Ca2+,but also through specific interactions with calreticulin and phospholipase Cδ1.
Determining the Kinetic Advantage to Mitochondrial Respiratory Supercomplex Formation. Cellular respiration in the mitochondria of higher organisms is paramount to supplying the ATP needed to sustain biological processes. The redox reactions of the mitochondrial respiratory chain, which consists of four transmembrane enzyme complexes, a lipid-soluble electron carrier (ubiquinone), and a water-soluble electron carrier (cytochromec), generate the electrochemical proton gradient necessary to drive ATP synthesis. While ample evidence has shown that these enzymes exist both as solitary units and supercomplex assemblies in the inner mitochondrial membrane,controversy surrounds the functional and kinetic advantage gained by the formation of the mutli-enzyme supercomplexes. To aid in resolving the controversy, we have an initiated an in-depth steady-state and pre-steady state kinetic analysis of the enzyme activity of intact mitochondria to determine if there is any kinetic or regulatory advantage to complex formation. Our future goals will also use novel biophysical techniques to probe the nature of the transient protein interactions governing supercomplex formation in both physiological and pathological 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 vitro techniques 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 enzymology, include protein engineering, site-directed mutagenesis, steady-state and pre-steady-state kinetics, FRET, and 1D/2D NMR.
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., Sereeruk, 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 of Rhizobium 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 the Rhizobium 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 transferase domain mechanism of pyruvate carboxylase from Rhizobium etli. Biochemistry, 48, 4305-4313. PMID: 19341298
View complete PubMed listing for Zeczycki TN
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