Fine tuning of the catalytic properties of carbonic anhydrase. Studies of a Thr200----His variant of human isoenzyme II.
The active sites of carbonic anhydrases I contain a unique histidine residue at sequence position 200. To test the hypothesis that His200 is essential for the isoenzyme-specific catalytic and inhibitor-binding properties of carbonic anhydrases I, a variant of human carbonic anhydrase II, having His200 for Thr200, was prepared by oligonucleotide-directed mutagenesis. The variant has a circular dichroic spectrum that is indistinguishable from that of the parent enzyme. The kinetics of CO2 hydration and HCO3- dehydration has been investigated. The results show that the amino acid substitution has led to changes of catalytic parameters as well as Ki values for anion inhibition in the expected directions towards the values for isoenzyme I. However, the maximal 4-nitrophenyl acetate hydrolase activity of the variant is higher than for any naturally occurring carbonic anhydrase studied so far. A detailed analysis of the kinetic observations suggests that the modification has resulted in a change of the step that limits the maximal rate of CO2 hydration at saturating buffer concentrations. This rate-limiting step is an intramolecular proton transfer in unmodified isoenzyme II and, presumably, HCO3- dissociation in the variant and in human isoenzyme I. A free-energy profile for the dominating pathway of CO2 hydration at high pH was constructed. The results suggest that the major effect of His200 is a stabilization of the enzyme-HCO3- complex by about 7.5 kJ/mol (variant) and 6.1 kJ/mol (human isoenzyme I) relative to unmodified isoenzyme II, while proton transfer between the metal site and the reaction medium is only marginally affected by the amino acid replacement.
Behravan
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Avdelningen för biokemi, Umeå Universitet, Sweden.
Jonsson
B H
BH
Lindskog
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D000838:Anions / Q000494:pharmacology; D001665:Binding Sites; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology*; D006639:Histidine / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D013329:Structure-Activity Relationship; D013912:Threonine / Q000502:physiology
D000838:Anions; D002257:Carbonic Anhydrase Inhibitors; D013912:Threonine; D006639:Histidine; D002256:Carbonic Anhydrases