Enzyme kinetics in solvents of increased viscosity. Dynamic aspects of carbonic anhydrase catalysis.
The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten parameters associated with CO2 hydration and HCO3- dehydration were deduced and analyzed by utilizing a spectrophotometric stopped-flow technique. It was found that both kcatHCO3 (= 3.9 X 10(5) s-1 at pH 5.90) and kcatCO2 (= 1.2 X 10(5) s-1 at pH 5.90 and 8.6 X 10(5) s-1 at pH 8.80) steadily decreased with the addition of monomeric viscogen while both KmHC03- (= 20 mM at pH 5.90) and KmCO2 (= 18 mM at pH 5.90 and 13 mM at pH 8.80) remained independent of viscosity, within experimental error. These results indicate that some kind of proton-transfer-related event is primarily responsible for the observed rate decrease. The three polyhydroxy cosolutes exhibited significant differences with regard to the magnitude of the viscosity effect on the kcat of the enzyme, with glycerol affecting the largest decrease, sucrose affecting a moderate one, and ficoll having virtually no effect. The discrepancy between glycerol and sucrose could be largely reconciled by correcting for diffusion-unrelated effects as estimated from rate studies of considerably slower CA II catalyzed acetaldehyde hydration and p-nitrophenyl acetate hydrolysis. Ficoll, however, was found to be unsuitable as a viscogenic probe because it failed to appreciably decrease the mobilities of smaller ions (as deduced from electrolytic conductance measurements) despite its capacity to greatly increase the macroscopic viscosity of the medium. Our best estimates indicate that this reaction comes within ca. 30% of the diffusion limit at 0.890 cP and 25 degrees C for both CO2 hydration and HCO3- dehydration reactions. However, it is reasonable to expect this value to be considerably higher in the natural environment of the enzyme because of the relatively high viscosities attained in the interior of erythrocytes.
D000079:Acetaldehyde; D000818:Animals; D002021:Buffers; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D005362:Ficoll / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D013395:Sucrose / Q000494:pharmacology; D014783:Viscosity
D002021:Buffers; D007202:Indicators and Reagents; D005362:Ficoll; D013395:Sucrose; D002256:Carbonic Anhydrases; D000079:Acetaldehyde; D005990:Glycerol