title: The role of electrostatic interactions in protease surface diffusion and the consequence for interfacial biocatalysis.
abstract: This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, United States.
authors.forename: Bob E
authors.initials: BE
authors.lastname: Feller
authors.forename: James T
authors.initials: JT
authors.lastname: Kellis
authors.forename: Luis G
authors.initials: LG
authors.lastname: Cascão-Pereira
authors.forename: Channing R
authors.initials: CR
authors.lastname: Robertson
authors.forename: Curtis W
authors.initials: CW
authors.lastname: Frank
mesh_terms: D000327:Adsorption; D000818:Animals; D055162:Biocatalysis; D002417:Cattle; D040143:Cellulomonas / Q000201:enzymology; D004058:Diffusion; D007700:Kinetics; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity; D013499:Surface Properties
chemical_list: D010447:Peptide Hydrolases