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

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• 作者：Douglas B. Craig ; ^{d.craig@uwinnipeg.ca} ; Steffany D. King ; Gundars Reinfelds ; Anna R.P. Henderson ; Tabitha E.H. Wood
• 关键词：Single molecule ; β-glucuronidase ; Catalytic rate ; Electrophoretic mobility ; Activation energy ; Heterogeneity ; Capillary electrophoresis
• 刊名：International Journal of Biological Macromolecules
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：96
• 期：Complete
• 页码：669-674
• 全文大小：819 K
• 卷排序：96

文摘

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 ± 0.1 m2V−1 s−1 (N = 49) with a total range of −0.6 to −1.3 × 10−8 m2V−1 s−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 ± 19,000 min−1 (N = 49) with a total range of 14,000 to 130,000 min−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 ± 18 kJ mol−1 (N = 7) with a total range of 18–66 kJ mol−1.