Electrophoretic mobility, catalytic rate, and activation energy of catalysis of single molecules of the enzyme β-glucuronidase from Escherichia coli

• Published in: International journal of biological macromolecules 2017-03, Vol.96, p.669-674
• Main Authors: Craig, Douglas B., King, Steffany D., Reinfelds, Gundars, Henderson, Anna R.P., Wood, Tabitha E.H.
• Format: Article
• Language: English
• Subjects: Activation energy; beta-Galactosidase - isolation & purification; beta-Galactosidase - metabolism; Biocatalysis; Capillary electrophoresis; Catalytic rate; Electrophoresis, Capillary; Electrophoretic mobility; Enzyme Activation; Escherichia coli - enzymology; Heterogeneity; Kinetics; Single molecule; β-glucuronidase
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2016.12.034

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.