title: Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme β-glucuronidase from Escherichia coli.
abstract: Single molecule assays were performed on the enzyme E. coli β-glucuronidase using a capillary electrophoresis-based protocol. Electrophoretic mobility, catalytic rate and activation energy of catalysis were all found to be heterogeneous. The average mobility at 22°C was -1.1×10-8±0.1m2V-1s-1(N=49) with a total range of -0.6 to -1.3×10-8m2V-1s-1. The range in electrophoretic mobility suggests that the differences in shape or charge of the individual molecules underlying the heterogeneity are likely minimal. The average catalytic rate at 22°C was 37,000±19,000min-1(N=49) with a total range of 14,000 to 130,000min-1. Both of these properties were measured simultaneously for each of the molecules. There was a weak correlation (r2=0.43) between mobility and rate with the molecules with a less negative mobility having a tendency to have a higher rate. The average activation energy of catalysis, as determined by comparing rates at 22 and 35°C, was found to be 48±18kJmol-1(N=7) with a total range of 18-66kJmol-1.
authors.affiliation: Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada. Electronic address: d.craig@uwinnipeg.ca.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Craig
authors.affiliation: Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
authors.forename: Steffany D
authors.initials: SD
authors.lastname: King
authors.affiliation: Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
authors.forename: Gundars
authors.initials: G
authors.lastname: Reinfelds
authors.affiliation: Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
authors.forename: Anna R P
authors.initials: ARP
authors.lastname: Henderson
authors.affiliation: Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
authors.forename: Tabitha E H
authors.initials: TEH
authors.lastname: Wood
mesh_terms: D055162:Biocatalysis; D019075:Electrophoresis, Capillary; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D001616:beta-Galactosidase
keywords: Activation energy; Capillary electrophoresis; Catalytic rate; Electrophoretic mobility; Heterogeneity; Single molecule; β-glucuronidase