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Deoxy and deoxyfluoro glycosides as mechanistic probes of Escherichia coli (lacZ) B-galactosidase

University of British Columbia

The mechanism of galactoside hydrolysis by Escherichia coli (lacZ) β-galactosidase was probed with a series of 2',4' dinitrophenyl deoxy and deoxyfluoro glycopyranosides. A two-step mechanism has been proposed previously for this process: 1) cleavage of the glycosidic bond and formation of a covalent galactosyl-enzyme intermediate ("galactosylation"); 2) hydrolysis of the mtermediate to give free enzyme and galactose ("degalactyosylation"). A series of deoxy and deoxyfluoro analogs of 2',4'-dinitrophenyl-β,-D- galactopyranoside was prepared for this study. The 2-deoxyfluoro derivative was found to be an effective mechanism-based inactivator of E. coli (lacZ) β-galactosidase. This compound thus joins a class of 2-deoxy-2-fluoro glycosides which inactivate glycosidases of this type by the accumulation of a stable glycosyl-enzyme intermediate. The active site-directed nature of this inhibitor was shown by protection against inactivation by a competitive ligand and the near 1:1 stoichiometry of dinitrophenolate release with enzyme inactivation. Furthermore, when freed from excess inactivator the 2-deoxy-2-fluorogalactosyl enzyme intermediate turned over slowly in buffer (t₁⃓₂ = 69 h at 25°C) and exhibited enhanced rates of reactivation in the presence of the acceptors methanol or glucose, providing strong evidence that the intermediate is catalytically competent The other deoxy and deoxyfluoro analogs synthesized were substrates for the enzyme although the rates of enzymic hydrolysis were two to four orders of magnitude slower than have been measured for the parent compound. These large rate reductions are thought to result primarily from the loss of important transition state binding interactions due to the substitution of a hydrogen or a fluorine for a hydroxyl at a given position on the galactopyranose ring. These results strongly suggest that much of the catalytic power of the enzyme is derived from non-covalent interactions between the enzyme active site and the galactopyranose ring of the substrate. A linear free energy relationship (r = 0.80) was shown to exist between the logarithm of kcat/Km for the enzyme-catalyzed reaction and the logarithm of the first order rate constant for the spontaneous hydrolysis of the same series of deoxy and deoxyfluoro glycopyranosides. Since the spontaneous process has considerable oxocarbonium ion character at the transition state, these data suggest that the enzymic mechanism involves a similar electron deficient transition state.

Text

2010-11-22

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http://hdl.handle.net/2429/30083

Chemistry

University of British Columbia

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