Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B.
Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.
D000086:Acetazolamide / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D002256:Carbonic Anhydrases / Q000032:analysis / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008027:Light; D008623:Mercaptoethanol / Q000494:pharmacology; D009578:Nitrobenzenes; D010084:Oxidation-Reduction; D010777:Photochemistry; D013997:Time Factors
D000596:Amino Acids; D009578:Nitrobenzenes; D006639:Histidine; D008623:Mercaptoethanol; D004950:Esterases; D002256:Carbonic Anhydrases; D000086:Acetazolamide