The mechanism of glucoside hydrolysis by Agrobacterium β-glucosidase has been investigated through the study of linear free energy relationships and a-secondary deuterium kinetic isotope effects. A two-step mechanism has previously been proposed for this process, consisting of;
(1) cleavage of the glucosidic bond and formation of a covalent glucosyl-enzyme
intermediate ("glucosylation"), and
(2) hydrolysis of the intermediate to yield free enzyme and glucose
("deglucosylation").
Values of kcat and Km were determined for enzymic hydrolysis of fifteen substituted phenyl β-D-glucopyranosides with leaving group pKa's ranging from 3.96 to 10.34. A linear free energy correlation of log(kcat) vs. leaving group pKa resulted in a concave-downward plot with a break near pKa 8, indicating a change in rate-determining step of a multistep reaction. The rates of hydrolysis of substrates with leaving group pKa's < 8 are independent of phenol structure, indicating that deglucosylation is rate-limiting. Glucosides with leaving group pKa's > 8 exhibit a linear dependence of hydrolysis rate upon pKa, and thus it is proposed that glucosylation is the rate-determining step for these substrates. The value of the Hammett reaction constant, ρ, is 1.6, indicating that cleavage of the glucosidic bond is significantly advanced at the transition state.
The α-secondary deuterium kinetic isotope effects on hydrolysis of five substituted phenyl β-D-glucopyranosides were determined (deuterium substitution at the anomeric center), and the values were found to be segregated into two groups. The faster substrates (leaving group pKa < 8) exhibited kH/kD values of approximately 1.11, while values for the slower substrates averaged 1.06. These results support the hypothesis of a change in rate-determining step as leaving group pKa decreases. The magnitude of the isotope effect on glucosylation indicates a small amount of sp³ to sp² rehybridization at the transitionstate, which combined with the ρ value for this process suggests a substantial degree of nucleophilic participation of the enzymic carboxylate.
2-Deoxy-2-fluoro-D-glucosides with highly activated leaving groups are potent covalent inactivators of Agrobacterium β-glucosidase which operate by trapping the
enzyme as its glucosyl-enzyme intermediate. Values of ki and Ki were determined for six substituted phenyl 2-deoxy-2-fluoro-β-D-glucopyranosides whose leaving group pKa's
ranged from 3.96 to 7.18. A linear correlation was observed for both log(ki) and log(ki/Ki) vs. leaving group pKa, with ρ values of 2.0 and 2.7, respectively, which
indicates that cleavage of the glycosidic bond is virtually complete at the transition state. Such an observation of a linear free energy relationship between the rate of enzyme inactivation and the electronic structure of the inactivator is rarely accomplished in enzymology. Preliminary investigation of the α-secondary deuterium kinetic isotope effect on enzyme inactivation by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside indicates that the effect is quite small, probably 0-5%. These results suggest that the inactivation proceeds via an essentially concerted mechanism and that the transition state has little oxocarbonium ion character. / Science, Faculty of / Chemistry, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/29141 |
| Date | January 1990 |
| Creators | Kempton, Julie B. |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
Page generated in 0.0121 seconds