The kinetics of both the association and dissociation reaction of two heterocyclic sulfonamide inhibitors (S), methazolamide and ethoxzolamide, with human carbonic anhydrase (carbonate hdyro-lyase, EC 22.214.171.124) isoenzyme B (CA) has been studied as a function of pH, using fluorescence stopped-flow instrumentation. The dissociation rate constants are similar for the two inhibitors and are unaffected by pH changes in the range 6.0-10.0. The association rate constants exhibit bell-shaped curves as a function of pH, consistent with the rate being controlled by two ionizing groups. The evidence suggests that one is an ionizing group on the enzyme with a pK near 7.5, which controls the low-pH limb of the curve, while the other function, controlling the high-pH limb, is the ionization of the sulfonamide group of the inhibitor. The data are consistent with the enzyme-drug reaction being either CA + SH or CAH+ + S-.
ACKNOWLEDGMENT We wish to thank Mr. P. C. Turner for expert technical assistance.
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