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Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase.

ABSTRACT: Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2',4'-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.

SUBMITTER: McCarter JD

PROVIDER: S-EPMC1132963 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Project description:1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated. The kcat. values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The kcat. values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates. Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol. Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga. beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes. Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change. Values of kcat. for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar. The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.

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Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase.

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