Fine tuning of the catalytic properties of human carbonic anhydrase II

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Abstract
The active-site residue Thr200 in human carbonic anhydrase II has been replaced by several different amino acids by site-directed mutagenesis. The CO2 hydration and 4-nitrophenyl acetate hydrolase activities of these variants have been measured, as well as inhibition by the monovalent anion, SCN−. The results show that the replacement of Thr200 with Ser or Ala has no significant effect on the catalyzed rates of CO2 hydration. Also, variants with Asn200 and Gly200 have high activities, whereas the activities of variants with Val, Ile or Arg at position 200 are reduced by factors of 2–3 compared to the unmodified enzyme. The variant with Asp200 has a very low activity in both reactions studied, while most of the other variants have enhanced esterase activities, Thr200↔ Arg isoenzyme II as much as sevenfold. The Asp200 variant has a low affinity for SCN− as well as for a sulfonamide inhibitor, whereas all the other variants bind SCN− more strongly than unmodified enzyme. While His200 characterizes carbonic anhydrases I, the presence of Arg, Val or Ile as well as His at position 200 in human isoenzyme II seems to result in isoenzyme-I-like functional properties.