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J. Biol. Chem., Vol. 260, Issue 6, 3484-3489, Mar, 1985
T Kararli and DN Silverman
Using stopped flow methods, we have measured the steady state rate
constants and the inhibition by N3- and I- of the hydration of CO2
catalyzed by carbonic anhydrase III from cat muscle. Also, using
fluorescence quenching of the enzyme at 330 nm, we have measured the
binding of the sulfonamide chlorzolamide to cat carbonic anhydrase III.
Inhibition by the anions was uncompetitive at pH 6.0 and was mixed at
higher values of pH. The inhibition constant of azide was independent of pH
between 6.0 and 7.5 with a value of KIintercept = 2 X 10(-5) M; the binding
constant of chlorzolamide to cat carbonic anhydrase III was also
independent of pH in the range of 6.0 to 7.5 with a value Kdiss = 2 X
10(-6) M. Both of these values increased as pH increased above 8. There was
a competition between chlorzolamide and the anions N-3 and OCN- for binding
sites on cat carbonic anhydrase III. The pH profiles for the kinetic
constants and the uncompetitive inhibition at pH 6.0 can be explained by an
activity-controlling group in cat carbonic anhydrase III with a pKa less
than 6. Moreover, the data suggest that like isozyme II, cat isozyme III is
limited in rate by a step occurring outside the actual interconversion of
CO2 and HCO3- and involving a change in bonding to hydrogen exchangeable
with solvent water.
Inhibition of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle
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