Catalytic enhancement of human carbonic anhydrase III by replacement of phenylalanine-198 with leucine.
Carbonic anhydrase III, a cytosolic enzyme found predominantly in skeletal muscle, has a turnover rate for CO2 hydration 500-fold lower and a KI for inhibition by acetazolamide 700-fold higher (at pH 7.2) than those of red cell carbonic anhydrase II. Mutants of human carbonic anhydrase III were made by replacing three residues near the active site with amino acids known to be at the corresponding positions in isozyme II (Lys-64----His, Arg-67----Asn, and Phe-198----Leu). Catalytic properties were measured by stopped-flow spectrophotometry and 18O exchange between CO2 and water using mass spectrometry. The triple mutant of isozyme III had a turnover rate for CO2 hydration 500-fold higher than wild-type carbonic anhydrase III. The binding constants, KI, for sulfonamide inhibitors of the mutants containing Leu-198 were comparable to those of carbonic anhydrase II. The mutations at residues 64, 67, and 198 were catalytically independent; the lowered energy barrier for the triple mutant was the sum of the energy changes for each of the single mutants. Moreover, the triple mutant of isozyme III catalyzed the hydrolysis of 4-nitrophenyl acetate with a specific activity and pH dependence similar to those of isozyme II. Phe-198 is thus a major contributor to the low CO2 hydration activity, the weak binding of acetazolamide, and the low pKa of the zinc-bound water in carbonic anhydrase III. Intramolecular proton transfer involving His-64 was necessary for maximal turnover.
LoGrasso
P V
PV
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610.
Tu
C K
CK
Jewell
D A
DA
Wynns
G C
GC
Laipis
P J
PJ
Silverman
D N
DN
D001639:Bicarbonates / Q000737:chemistry; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D010649:Phenylalanine / Q000235:genetics
D001639:Bicarbonates; D010103:Oxygen Isotopes; D010649:Phenylalanine; D002256:Carbonic Anhydrases; D007930:Leucine