PMID:7758465

Citation	Engstrand, C, Jonsson, BH and Lindskog, S (1995) Catalytic and inhibitor-binding properties of some active-site mutants of human carbonic anhydrase I. Eur. J. Biochem. 229:696-702
Abstract	Three isozyme-specific residues in the active site of human carbonic anhydrase I, Val62, His67, and His200, have been changed by site-directed mutagenesis to their counterparts in human carbonic anhydrase II, Asn62, Asn67, and Thr200. A double mutant, containing Asn62 and Asn67, and a triple mutant, containing all three alterations, were also produced. The rates of CO2 hydration and ester hydrolysis catalyzed by these mutants, the inhibition of these enzymes by the anions, SCN-, and I-, and the binding of the sulfonamide inhibitors, dansylamide and MK-417 (a thienothiopyran-2-sulfonamide) have been measured. The results suggest that the effect of His200 in isozyme I is to prolong the lifetime of the enzyme-bicarbonate complex and to increase the pKa of the catalytic group, a zinc-coordinated water molecule. For isozyme I, Val62 and His67 might interfere with the function of a proton 'shuttle' group in the active site, thus maintaining the buffer specificity of a compulsory proton-transfer step. The single mutations have small effects on anion binding. Only the triple mutant has anion-binding properties resembling those of isozyme II. All mutants show altered sulfonamide-binding properties. In particular, the binding specificity is affected. While wild-type isozyme I binds dansylamide 50 times more strongly than MK-417, the triple mutant shows a reversed selectivity and binds MK-417 nearly 50 times more strongly than dansylamide.
Links	PubMed
Keywords	Amino Acids/chemistry; Binding Sites; Carbonic Anhydrase Inhibitors/metabolism; Carbonic Anhydrases/chemistry; Carbonic Anhydrases/genetics; Carbonic Anhydrases/metabolism; Catalysis; Humans; Isoenzymes/chemistry; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; Magnetic Resonance Spectroscopy; Mutagenesis, Site-Directed; Mutation; Structure-Activity Relationship
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:CAH2		GO:0004089: carbonate dehydratase activity	ECO:0000315:		F		Table 2	complete CACAO 5529
HUMAN:CAH2	enables	GO:0004089: carbonate dehydratase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:CAH1		GO:0004089: carbonate dehydratase activity	ECO:0000315:		F		Table 1	complete CACAO 5434
HUMAN:CAH1		GO:0004064: hydrolase activity	ECO:0000315:		F		Table 4	complete CACAO 5436
HUMAN:CAH1		GO:0004064: arylesterase activity	ECO:0000315:		F		Table 4	complete CACAO 6167
HUMAN:CAH1	enables	GO:0004064: arylesterase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:CAH1	enables	GO:0004089: carbonate dehydratase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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