The Interaction of Sulfate with Carbonic Anhydrase

In the absence of sulfate the pH/rate profile for the 4‐nitrophenyl acetate hydrolase activity of bovine carbonic anhydrase is complex. The results fit with a microscopic ionization scheme involving two electrostatically interacting groups. The activity depends on the concentration of the basic form of one of these groups. Proton NMR spectra show that the active site residue, His‐64, has titration behaviour corresponding to that of the second group of the ionization scheme. The addition of increasing concentrations of Na2SO4 gradually converts the pH/rate profile to that of a simple titration curve. A pKa of 6.9 is found at 50 mM Na2SO4. Concomitantly the titration curve of His‐64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO2−4binding occurs only when both His‐64 and the activity‐linked group are protonated. At constant pH, sulfate behaves as if it inhibits the enzyme only partially. However, data are presented suggesting that the enzyme‐sulfate complex is inactive, but the binding of SO2−4depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensat