An Inverse Michaelis-Menten Approach for Interfacial Enzyme Kinetics

Interfacial enzyme reactions are ubiquitous both in vivo and in technical applications, but analysis of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis-Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive experimental study of the enzymatic hydrolysis of insoluble cellulose indeed showed that the conventional approach had a limited applicability. Instead we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite experimental limit, where the concentration of enzyme far exceeded the molar concentration of accessible surface sites. Under this condition, an inverse Michaelis-Menten equation, where the roles of enzyme and substrate had been swapped, proved to be readily applicable. We suggest that this inverted approach provides a general tool for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis-Menten analysis reveals the density of enzyme attack sites on the substrate surface as probed by one specific enzyme. This density, which is an analogue to a molar substrate concentration for interfacial reactions, was shown to vary strongly even among related enzymes. This difference reflects how the enzyme discriminates between local differences in surface structure on the substrate.