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 Na 2 SO 4 gradually converts the pH/rate profile to that of a simple titration curve. A p K a of 6.9 is found at 50 mM Na 2 SO 4 . Concomitantly the titration curve of His‐64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO 2− 4 binding 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 SO 2− 4 depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensated by a decrease of the binding affinity. The binding of a monovalent anion, C1 − , also depends on ionic strength, but this dependence is much less prominent than for SO 2‐ 4 . Sulfate shows non‐competitive behaviour with respect to CO 2 and appears to compete with HCO − 3 . Iodide and sulfate appear to be mutually competitive. The low pH limit of K i for I − is 3 mM and about 0.1 mM in the presence of 50 mM Na 2 SO 4 and in the absence of sulfate respectively. In the absence of sulfate the Co(II)‐substituted bovine enzyme shows a complex pH/rate profile similar to that of the native enzyme. Changes of the optical spectrum parallel those of the activity. Relaxation rates (T −1 1 )of water protons in the presence of Co(II) enzyme indicate that the Co(II)ion might have a H 2 O ligand at low pH in sulfate‐free solutions. The effects on sulfate of human carbonic anhydrase II (or C) are similar to those on the bovine enzyme. The effects of sulfate on the human 1 (or B) enzyme are smaller.