A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.
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)
Selwood
T
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Department of Organic Chemistry, University of Bristol, U.K.
Sinnott
M L
ML
D002384:Catalysis; D003903:Deuterium; D004140:Dinitrophenols / Q000378:metabolism; D004147:Dioxins / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism; D005696:Galactosidases / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007763:Lac Operon; D008274:Magnesium / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D013379:Substrate Specificity; D014316:Tritium; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D004140:Dinitrophenols; D004147:Dioxins; D005960:Glucosides; D014316:Tritium; C025193:4-nitrophenyl beta-D-glucoside; C064737:3,4-dinitrophenyl-beta-galactopyranoside; D003903:Deuterium; D005696:Galactosidases; D001616:beta-Galactosidase; D008274:Magnesium; D005690:Galactose; D000432:Methanol