| pubmed-article:8485128 | pubmed:abstractText | The functional importance and structural determinants of a conserved hydrophobic pocket in human carbonic anhydrase II (CA II) were probed by preparing and characterizing 13 amino acid substitutions at Leu-198, situated at the mouth of the pocket. The pH dependence of the esterase activity reveals that activity decreases (up to 120-fold) as the amino acid size and charge at position 198 are varied while the pKa of the zinc-bound water molecule increases (up to 1 pH unit). Intriguingly, the pH dependence of the Leu-198-->Glu substitution is parabolic (pKas approximately 6 and 9), consistent with introduction of a general base-catalyzed mechanism. Kinetic characterization of CO2/HCO3- interconversion catalyzed by four variants (Leu-198-->Ala, His, Arg, and Glu) reveals that increasing the size of the hydrophobic pocket (Ala) does not compromise catalysis (approximately 3-fold decrease); however, substitution of charged (Arg and Glu) and larger (His) amino acids decreases kcat/KM for CO2 hydration substantially (17-fold, 19-fold, and 10-fold, respectively) but not completely. log kcat/KM for CO2 hydration, HCO3- dehydration, and p-nitrophenyl acetate hydrolysis correlates with the hydrophobicity of the residue at 198, likely reflecting desolvation or electrostatic destabilization of the ground state. The X-ray crystal structures of the Leu-198-->His, Glu, and Arg variants (Nair & Christianson, 1993) indicate that the His and Glu side chains are accommodated by minor structural reorganization leading to a wider mouth for the hydrophobic pocket while the Arg side chain blocks the pocket. Infrared spectroscopy of CO2 bound to either wild-type CA II or the Leu-198-->Arg variant indicates that the Arg substitution both decreases the affinity and alters the position of CO2 binding, suggesting that the hydrophobic pocket forms the CO2 binding site in CA II. Finally, a 1.5-fold increase (Leu-198-->Ala) and 12-fold decrease (Leu-198-->Arg) in kcat for CO2 hydration, indicative of the rate constant for intramolecular proton transfer from zinc-bound water to His-64, are likely mediated by changes in the active site solvent structure. | lld:pubmed |
