The unifying goal of this proposed research is to investigate the properties of water as a ligand of metals in metalloproteins and to elucidate the role of that water in catalysis by some selected metalloenzymes, especially carbonic anhydrase. I) The hydrogen-deuterium fractionation factor for water as a ligand of metals will be determined by measuring the NMR relaxation enhancement of the protons of water in the presence of complexes and enzymes containing paramagnetic metals. The fractionation factor of water exchanging from the coordination sphere of cobalt in Co(II)-substituted isozymes of carbonic anhydrase and Co(II)-alcohol dehydrogenase will be determined. This information will be used to help interpret observed isotope effects and to arrive at a better understanding of the mechanisms. II) The steady state and 180 exchange kinetics for catalysis by the newly-discovered carbonic anhydrase III from mammalian muscle will be determined. This includes the rate of release from the active site of water bearing substrate oxygen. Of primary importance is to find the pK's of activity-controlling groups since preliminary evidence indicates activity independent of pH from 5 to 8, in strong contrast to the other isozymes. III) A comparison of the kinetics of the three isozymes of mammalian carbonic anhydrase will provide a good opportunity to learn which factors influence the catalysis and the rate of release of water from the active site. The working hypothesis to be tested is that Lys 64 and Arg 67 in the active-site cleft of bovine carbonic anhydrase III cause low activity and low rate of release of water. To test this, chemical modifications will be made of these residues. A cloned gene for mouse, red cell carbonic anhydrase II placed in E. coli will serve as a source of isozyme II for NMR studies of 13C and 15N labeled enzyme. Chemical shifts of these resonances will be measured in the presence of substrate and inhibitors to detect interactions and deduce the role of active-site residues in the catalytic pathway.
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