A steady-state kinetic investigation of the uricase reaction mechanism

The steady-state kinetics of porcine liver uricase has been investigated over an extensive range of both urate and oxygen concentration. Spectrophotometric assay of the accumulation of reaction intermediate which absorbs strongly in the ultraviolet region as well as the oxygen electrode were used to minimize inaccuracies in initial velocity measurements. The Superoxide radical could not be detected as a product of the uricase reaction. Reciprocal plots with both urate and oxygen showed extensive nonlinearity. Mechanisms involving second binding sites on uricase appear unlikely in view of the structural properties of the enzyme. A random substrate binding scheme has been shown to be consistent with all of the nonlinear reciprocal plots and is thus suggested as the most probable mechanism of action for uricase.