title: Determination of kinetic parameters for interfacial enzymatic reactions on self-assembled monolayers.
abstract: This paper reports a method to characterize the kinetic constants for the action of enzymes on immobilized substrates. This example uses cutinase, a serine esterase that hydrolyzes 4-hydroxyphenyl valerate moieties that are immobilized on a self-assembled monolayer of alkanethiolates on gold. The product of the enzyme reaction is a hydroquinone, which is redox active and therefore permits the use of cyclic voltammetry to monitor the extent of reaction in situ. A kinetic model based on the Michaelis-Menten formalism is used to analyze the dependence of initial rates of reaction on both the substrate density and the enzyme concentration. The resulting value of k(cat)/K(M) for the interfacial reaction is comparable to that for a homogeneous phase reaction with a substrate of similar structure. This strategy of using monolayers presenting substrates for the enzyme and cyclic voltammetry to measure reaction rates provides quantitative and real-time information on reaction rates and permits a level of analysis of interfacial enzyme reactions that to date has been difficult to realize.
authors.affiliation: Department of Chemistry, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Satish
authors.initials: S
authors.lastname: Nayak
authors.forename: Woon-Seok
authors.initials: WS
authors.lastname: Yeo
authors.forename: Milan
authors.initials: M
authors.lastname: Mrksich
mesh_terms: D004563:Electrochemistry; D004800:Enzymes, Immobilized; D004950:Esterases / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014631:Valerates / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D014631:Valerates; D006046:Gold; D004950:Esterases; C022635:serine esterase