Department of Biochemistry and Molecular Biology My labs current interests are in the general area of enzyme structure, function and mechanism. Of particular interest to the lab is the role that conformational dynamics play in enzyme mechanism and ligand recognition. We are currently investigating these phenomena in the important metabolic enzyme phosphoenolpyruvate carboxykinase using primarily enzyme kinetics, x-ray crystallography and other biophysical approaches. Our hope is that the information gained from these studies will not only lend further insight into how nature's catalysts are able to carry out difficult chemical transformations but will also aid in the development of effective inhibitors of PEPCK as potential anti-hyperglycemic agents in the treatment of diabetes.
An additional area of research in my lab is focused upon determining the mechanisms of catalysis and substrate selectivity in families of highly selective proteases.
1) Kex2/Pro-protein convertase family
Members of this family are known virulence factors in pathogenic fungi, and homologues in higher eukaryotes play important roles not only in homeostatic functions but also in disease states including cancer, Alzheimer’s and anthrax toxicity. With the observation of both redundant and non-redundant activities of these enzymes in vivo we are interested in structurally and mechanistically characterizing this family of proteases to determine the structural determinants of their high degree of selectivity for their biologically and medically important protein substrates.
2) IgA protease family
IgA proteases are a family of secreted proteases that are produced by pathogenic strains of Haemophilus, Neisseria and Streptococcus that are the causative agents of such diseases as gonorrhea and bacterial meningitis. These enzymes cleave human secretory IgA1 in the hinge region of the heavy chain, inactivating the molecule and allowing the bacteria to circumvent host defenses mediated by IgA1 in the mucosal linings. We are very interested in the extreme substrate selectivity exhibited by this family of enzymes, which includes both serine- and metallo-endoproteases.
Sullivan, S.M. and Holyoak, T. Structures of rat cytosolic PEPCK: Insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid. Biochemistry, 2007; 46, 10078-88.
Wheatley, J.L. and Holyoak, T. Differential P1 arginine and lysine recognition in the protypical proprotein convertase Kex2. Proc Natl Acad Sci USA 2007; 104, 6626-31.
Holyoak, T., Sullivan, S.M. and Nowak, T. Structural insights into the mechanism of PEPCK catalysis. Biochemistry 2006; 45, 8254-63.
Williams, R., Holyoak, T., McDonald, G., Gui, C., and Fenton, A.W. Differentiating a ligand’s chemical requirements for allosteric interactions from those for protein binding; phenylalanine inhibition of pyruvate kinase. Biochemistry 2006; 45, 5421-29.
Fenn, T.D., Holyoak, T., Stamper, G.F. and Ringe, D. Effect of a Y265F mutant on the transamination based cycloserine inactivation of alanine racemase. Biochemistry 2005, 44, 5317-27.
Holyoak, T. and Nowak, T. pH dependence of the
reaction catalyzed by avian phosphoenolpyruvate
carboxykinase. Biochemistry 2004; 43, 7054-65.
Holyoak, T., Kettner, C. A., Petsko, G. A., Fuller, R. S., and Ringe, D. Structural basis for differences in selectivity in Kex2 and furin protein convertases. Biochemistry 2004; 43, 2412-21.
Holyoak, T., Fenn, T.D., Wilson, M.A., Moulin, A.G., Ringe, D. and Petsko, G.A. Malonate: A versatile cryo-protectant and stabilizing solution for salt grown macromolecular crystals. Acta. Cryst. 2003; D59, 2356-58.
Holyoak, T., Wilson, M. A., Fenn, T. D., Kettner, C. A., Petsko, G. A., Fuller, R. S., and Ringe, D. 2.4 angstrom resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor. Biochemistry 2003; 42, 6709-18.
Holyoak, T., and Nowak, T. Structural investigation of the
binding of nucleotide to phosphoenolpyruvate carboxykinase
by NMR. Biochemistry 2001; 40, 11037-47.
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