Kinetic Constants Determined from Membrane Transport Measurements: Carbonic Anhydrase Activity at High Concentrations

Abstract

Facilitated diffusion rates can be used to determine kinetic constants for rapid reactions occurring within membranes and thin fluid layers. We have applied this technique to the study of the reversible CO(2) hydration reactions catalyzed by carbonic anhydrase (EC 4.2.1.1; carbonate hydro-lyase). The experimental method entails the diffusion of tracer (14)CO(2) through Millipore filter membranes impregnated with aqueous bicarbonate solutions containing various concentrations of dissolved enzyme. A mathematical model of the simultaneous diffusion/reaction transport process is analyzed to predict the effective diffusion rate in terms of the relevant kinetic parameters. The solution to the mathematical model can be transformed to yield straight-line relations analogous to Lineweaver-Burk plots. The pseudo-first-order enzymatic rate constant for the hydration reaction can be determined from the slope or intercept of a plot of this straight-line relationship. Rate constants were accurately measured at high enzyme concentrations for reactions having half-times under a millisecond. The rate constants agree well with other reported kinetic constants for carbonic anhydrase, and the known pH-activity dependence and bicarbonate inhibition are quantitatively demonstrated. The specific activity is constant up to 4.0 mg/ml, which is believed to be the highest concentration at which the activity has been measured. The membrane transport technique has general applicability for other rapid reaction systems.