A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) β-galactosidase

Abstract

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)

Effect of an E461G Mutation of β-Galactosidase (Escherichia coli, lac Z) on pL Rate Profiles and Solvent Deuterium Isotope Effects

“…(1) The falloff in k cat /K m at high pH for wild-type ␤-galactosidase-catalyzed hydrolysis of Gal-OPNP observed in earlier work is consistent with a pK a of 8.3 for an essential amino-acid residue at the free enzyme (10). If this downward break is due to deprotonation of the carboxylic acid side chain of Glu 461, then a flat pH rate profile at high pH should be observed for the reaction catalyzed by the E461G enzyme.…”

“…The SDIE of (k cat ) HOH /(k cat ) DOD ϭ 1.7 for the wild-type enzyme catalyzed reaction shows that there is a significant decrease in the zero-point energy for a solventderived proton(s) that occurs on proceeding from the ES complex to the transition state for formation of the ␤-D-galactopyranosyl reaction intermediate (10). The decrease in the SDIE on k cat to 1.2 for the E461G enzyme-catalyzed reaction is consistent with the conclusion that the most of the SDIE on the wild-type enzyme-catalyzed reaction represents loss of zero-point energies of the carboxylic acid proton at a transition state in is partly transferred to the 4-nitrophenoxide ion leaving group.…”

“…(2) A solvent deuterium isotope effect (SDIE) of (k cat ) HOH /(k cat ) DOD ϭ 1.7 has been reported for wild-type ␤-galactosidase-catalyzed cleavage of Gal-OPNP (10). If this isotope effect results from loss of the zero-point energy for a solvent-derived proton at a transition state in which there is partial proton-transfer from the propionic acid side chain of Glu 461 to the oxygen leaving group, then removal of the proton from the transition state for the reaction catalyzed by E461G ␤-galactosidase should reduce the SDIE to around 1.0.…”

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…80 Similarly, solvent isotope effects on catalysis of pNPG suggest that proton transfer is part of the rate-limiting step for k cat but not for k cat /K m . 81 Proton transfer from Glu461 is expected to be involved in the chemical step for galactosylation but not for movement to the deep mode. These isotope effects can be at least partially explained by a rate-determining bond cleavage for k cat , and rate-determining progression to the deep mode for k cat /K m .…”

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

“…GH enzymes enhance the rate of this slow hydrolysis reaction using a general acid, usually the carboxylic acid of a Glu residue, which donates a proton to the leaving group oxygen in the rate-limiting step (8)(9)(10). Two models for the catalytic mechanism of β-glycoside hydrolases have been proposed: (i) a double displacement pathway first proposed by Koshland, essentially proceeding as an S N 2 reaction (11,12) and (ii) an oxocarbenium ion intermediate pathway originally proposed by Phillips (13), occurring as an S N 1 reaction (Fig.…”