Origin of viscosity effects in carbonic anhydrase catalysis. Kinetic studies with bulky buffers at limiting concentrations

In our earlier paper we showed that the rates of CO2 hydration and HCO3- dehydration catalyzed by the high-activity form of mammalian erythrocyte carbonic anhydrase (CA II) were dependent on solution viscosity increase and that the effect was linked to some kind of proton-transfer-related event Pocker, Y., & Janji?, N. (1987) Biochemistry 26, 2597-2606]. In order to further elucidate the source of the observed viscosity effect, the dependence of kcat and Km for CA II catalyzed HCO3- dehydration at pH 5.90 on sucrose-induced viscosity increase was investigated at several concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, including the very low buffer concentration region (less than 10 mM) where the proton transfer between the shuttle group on the enzyme and buffer becomes rate limiting. In all examined cases, kcat steadily decreased with added sucrose while Km remained independent of the viscosity increase. The extent to which this reaction was dependent on viscosity was found to be constant, within experimental error, over the entire range of MES buffer concentrations studied (1-20 mM). Furthermore, the viscosity effect was qualitatively and quantitatively the same when an exceptionally large buffer (i.e., bovine serum albumin) was used instead of the more commonly used biological buffer (i.e., MES).(ABSTRACT TRUNCATED AT 250 WORDS)