1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25°C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where Eo and Er represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k+4/k+2 is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k+4 and k+2 are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for Eo. This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.

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