Proton transfer and fast conformational changes in carbonic anhydrase sulfonamide complexes

Abstract

Laser photolysis and temperature-jump techniques were used to study the relaxation spectra of complexes of human carbonic anhydrase with aromatic sulfonamides. A conformational change was detected by both techniques, coupled to fast proton transfer processes. The rate of the conformational change is pH dependent and varies with the enzyme subforms between 200 μs and 1.8 ms at T = 25 °C. The effect was systematically investigated by temperature-jump techniques with different metal ions (Co, Mn, Cu, Ni) substituted for Zn at the active site. It is shown that the time constant of this conformational change at high pH is correlated to the esterase activity. This result supports the idea that an important feature of the Zn catalysis in this enzyme is the flexibility of the metal coordination sphere. For a faster time scale a laser photolysis apparatus was used to study the direct proton exchange between the coloured sulfonamide reporter group and the buffer molecules. Moreover, a very fast intramolecular proton transfer is shown to occur in deionized solutions of enzyme-inhibitor complexes at the active site, thus supporting the hypothesis of an essential role of this kind of process in the catalytic act.