Interactions of active-site residues and catalytic activity of human carbonic anhydrase III.

摘要: To elucidate the interactions between residues found in active-site cavity of human carbonic anhydrase III, we have prepared a series single and double mutants with Lys-64, Arg-67, Phe-198 replaced Ala, Asp, Glu, His, Leu. Rates catalysis were determined using 18O exchange CO2 water measured by mass spectrometry initial velocity stopped-flow spectrophotometry. Replacement these resulted increases kcat/Km for hydration as much 200-fold pKa zinc-bound 3.5 units. We conclude that effect replacements made at positions 64, 67, 198 general additive hydration, indicating there is no interaction sites affects catalytic interconversion HCO3-. One notable exception antagonism exhibited mutant III containing Glu-64 Leu-198. The data also show one source large enhancement Asp-198 presence both Lys-64; when Lys-64 was reduction activity observed. These results provide an additional view independent amino acids affect pathway isozyme least active known isozymes.