Interactions of active-site residues and catalytic activity of human carbonic anhydrase III.
To elucidate the interactions between residues found in the active-site cavity of human carbonic anhydrase III, we have prepared a series of single and double mutants with Lys-64, Arg-67, and Phe-198 replaced with Ala, Asp, Glu, His, and Leu. Rates of catalysis were determined using 18O exchange between CO2 and water measured by mass spectrometry and initial velocity measured by stopped-flow spectrophotometry. Replacement of these residues resulted in increases in kcat/Km for CO2 hydration as much as 200-fold and increases in the pKa of the zinc-bound water by as much as 3.5 units. We conclude that the effect of replacements made at positions 64, 67, and 198 were in general additive for kcat/Km for CO2 hydration, indicating that there is no interaction between these sites that affects the catalytic interconversion of CO2 and HCO3-. One notable exception is the antagonism exhibited by the double mutant of human carbonic anhydrase III containing Glu-64 and Leu-198. The data also show that one source of the large enhancement of kcat/Km for the mutant containing Asp-198 in human carbonic anhydrase III is the presence of both Asp-198 and Lys-64; when Lys-64 was replaced with Ala, a reduction of catalytic activity was observed. These results provide an additional view of the independent interactions of amino acids that affect the catalytic pathway of isozyme III, the least active of the known carbonic anhydrase isozymes.
Tu
C
C
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610-0267.
Chen
X
X
Ren
X
X
LoGrasso
P V
PV
Jewell
D A
DA
Laipis
P J
PJ
Silverman
D N
DN
D001665:Binding Sites; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D013816:Thermodynamics; D014867:Water; D015032:Zinc
D014867:Water; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases; D015032:Zinc