Influence of amino acid replacement at position 198 on catalytic properties of zinc-bound water in human carbonic anhydrase III.
Carbonic anhydrase III, found predominantly in skeletal muscle, is the least efficient of the mammalian carbonic anhydrases in catalyzing the hydration of CO2. Phenylalanine-198 is located on the hydrophobic side of the active-site cavity with its phenyl ring in the proximity of the catalytically active zinc-bound water. We replaced phenylalanine-198 in human carbonic anhydrase III with seven other amino acids (Ala, Asn, Asp, His, Leu, Tyr, Val) using site-directed mutagenesis. The catalytic properties of these enzymes were determined by stopped-flow spectrophotometry, and the exchange of 18O between CO2 and water was measured by mass spectrometry. All of the mutants had maximal values of kcat/Km for the hydration of CO2 enhanced, and five of the mutants had the pKa of the zinc-bound water increased compared with the wild-type enzyme. The largest effects were observed with the replacement Phe-198-->Asp which increased the maximal kcat/Km 140-fold and increased the pKa of the zinc-bound water from near 5 to 9.2. A Brønsted correlation was observed between log(kcat/Km) for hydration of CO2 and the pKa of the zinc-bound water (correlation coefficient r = 0.92); in addition, this pKa was inversely correlated with hydrophobicity of the residue at 198 (correlation coefficient r = -0.83). A direct correlation between the logarithm of the maximal kcat/Km for hydration and the logarithm of the pH-independent value of Ki for inhibition by cyanate (r = 0.95) indicated that the effect of the mutations at residue 198 occurred in large part by enhancement of the rate of dissociation of the enzyme-bicarbonate complex.
Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610.
P V
PV
LoGrasso
C
C
Tu
X
X
Chen
S
S
Taoka
P J
PJ
Laipis
D N
DN
Silverman
D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010649:Phenylalanine; D013431:Sulfates; D014867:Water / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D001639:Bicarbonates; D013431:Sulfates; D014867:Water; C012036:sodium sulfate; D002245:Carbon Dioxide; D010649:Phenylalanine; D002256:Carbonic Anhydrases; D015032:Zinc