This proposal outlines a research effort to delineate the roles of active site amino acids in human carbonic anhydrase II (CA II) in catalysis and drug (sulfonamide) binding. Sulfonamide inhibition of this enzyme has been used to treat glaucoma, epilepsy and ulcers. The effect of single-site mutations (prepared by oligonucleotide-directed mutagenesis) on CA II function, stability and structure will be measured. The goal of the proposed studies is to further our understanding of the importance of protein/substrate and protein/protein interactions for binding and catalysis. Experiments are designed to: 1) delineate the pathway and mechanism of CA II-catalyzed inter- and intramolecular proton transfer reactions; 2) measure the importance of general base catalysis or a proton shuttle mechanism in catalysis by CA II; 3) determine the effect of active site amino acids on the pKa of the enzyme-bound zinc-water ligand; 4) quantitatively evaluate the importance of hydrophobic interactions for binding and catalysis; 5) determine the effects of single amino acid changes on transition state structure; 6) quantitate the importance of isozymic active site variations; and 7) measure the effect of single site mutations on the structure of CA II using one- and two- dimensional NMR techniques. Information gained will impact on our understanding of the mechanism of carbonic anhydrases and other metalloenzymes, as well as the mechanism of proton transfers in all proteins. Quantitative dissection of the contribution of various residues to binding and catalysis for carbonic anhydrase hopefully can be generalized to other enzymes and will play a significant role in the design of active site inhibitors and new protein catalysts, as well as enlarge our understanding of enzymic catalysts.
| Gantt, Sister M Lucy; Decroos, Christophe; Lee, Matthew S et al. (2016) General Base-General Acid Catalysis in Human Histone Deacetylase 8. Biochemistry 55:820-32 |
| López, Jeffrey E; Sullivan, Eric D; Fierke, Carol A (2016) Metal-dependent Deacetylases: Cancer and Epigenetic Regulators. ACS Chem Biol 11:706-16 |
| Alam, Nawsad; Zimmerman, Lior; Wolfson, Noah A et al. (2016) Structure-Based Identification of HDAC8 Non-histone Substrates. Structure 24:458-68 |
| Dostál, Lubomír; Kohler, William M; Penner-Hahn, James E et al. (2015) Fibroblasts from long-lived rodent species exclude cadmium. J Gerontol A Biol Sci Med Sci 70:10-9 |
| Kim, Byungchul; Pithadia, Amit S; Fierke, Carol A (2015) Kinetics and thermodynamics of metal-binding to histone deacetylase 8. Protein Sci 24:354-65 |
| Olson, David E; Udeshi, Namrata D; Wolfson, Noah A et al. (2014) An unbiased approach to identify endogenous substrates of ""histone"" deacetylase 8. ACS Chem Biol 9:2210-6 |
| Wolfson, Noah A; Pitcairn, Carol Ann; Sullivan, Eric D et al. (2014) An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity. Anal Biochem 456:61-9 |
| Yang, Yue; Wang, Bing; Ucisik, Melek N et al. (2012) Insights into the mechanistic dichotomy of the protein farnesyltransferase peptide substrates CVIM and CVLS. J Am Chem Soc 134:820-3 |
| Zverina, Elaina A; Lamphear, Corissa L; Wright, Elia N et al. (2012) Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions. Curr Opin Chem Biol 16:544-52 |
| Hougland, James L; Gangopadhyay, Soumyashree A; Fierke, Carol A (2012) Expansion of protein farnesyltransferase specificity using ""tunable"" active site interactions: development of bioengineered prenylation pathways. J Biol Chem 287:38090-100 |
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