The carbonic anhydrases (CAs) catalyze a reaction of fundamental biochemical and physiological importance, the interconversion of carbon dioxide and bicarbonate ion CO2 + H2O - All CAs are zinc-dependent enzymes and a well-established mechanistic paradigm requires the coordination of substrate to the catalytic zinc ion (Zn2+). The structures determined for the ? class carbonic anhydrases (?-CAs), common in plants and bacteria, generally fall into two distinct subclasses based on the observed coordination of zinc. One subclass of ?-CAs coordinate Zn2+ tetrahedrally with four protein-derived ligands, and in this configuration access of substrate to the zinc coordination sphere is apparently blocked. The ability of substrate to coordinate to zinc is observed in the other structural subclass. Recent evidence supports the hypothesis that the blocked configuration, as seen for example in ECCA, a ?-CA from Escherichia coli, represents an inactive conformation of the enzyme, and that all such ?-CAs can undergo a transition to an active conformation. In addition, a unique, non-catalytic binding mode for the substrate bicarbonate was discovered in ECCA that appears to stabilize the blocked, inactive form of the enzyme and seems to represent a regulatory mechanism. This proposal specifically aims to characterize the allosteric bicarbonate site that is likely shared by many eubacterial ?-CAs, including a number of pathogens (e.g., Mycobacterium tuberculosis, Salmonella typhimurium). The structural and functional effects of its disruption by targeted mutagenesis are to be investigated by the primary method of X-ray crystallography, supported by kinetic measurements. In view of its potential as a site for therapeutic intervention, the characterization of the allosteric site will be furthered by a virtual screen for potential non-substrate ligands. Finally, the relationship between allosteric bicarbonate binding and the hypothesized structural transition in ECCA will be probed by testing the effects of mutations designed to shift the conformational equilibrium, The two observed structural subclasses serve as an explicit two-state model for regulation. This project will be attractive to students with interest in biochemistry, particularly protein structure and enzymology. It integrates a textbook example of an extremely fast enzyme with allosteric regulation of enzyme activity. The project also emphasizes computational methods, including molecular graphics, modeling, and informatics methods of drug discovery. This research will gather important basic information about eubacterial ?-carbonic anhydrases, enzymes known to be required for a number of pathological microorganisms. An allosteric ligand binding site and its effect on enzyme activity will be characterized with an eye to its development as a target of therapeutic drugs. ? ? ?
