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

Richard, John P.; Huber, R.E.; McCall, Deborah A.

doi:10.1006/bioo.2001.1206

Cited by 3 publications

(4 citation statements)

References 21 publications

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“…We conclude that Mg 2+ has an opposite stabilizing effect on the transition state for formation of H-1-E by cleavage of HO-1-OC 6 H 4 -4-NO 2 and a destabilizing effect on the transition state for hydrolysis of this intermediate. We are not able to offer an interpretation for this result, and note that the mechanism for Mg 2+ activation of β-galactosidase for hydrolysis of sugars is not well-understood (13,22,26,27,30,37,38).…”

Section: Hydrolysis Of 2-deoxy-β-d-galactopyranosylated Enzymementioning

confidence: 80%

Ground-State, Transition-State, and Metal-Cation Effects of the 2-Hydroxyl Group on β-d-Galactopyranosyl Transfer Catalyzed by β-Galactosidase (Escherichia coli, lac Z)

et al. 2005

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Substitution of the C2-OH group by C2-H at 4-nitrophenyl-beta-d-galactopyranoside to give 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside causes (1) a change in the rate-determining step for beta-galactosidase-catalyzed sugar hydrolysis from formation to breakdown of a covalent intermediate; (2) a 14 000-fold decrease in the second-order rate constant k(3)/K(d) for enzyme-catalyzed transfer of the beta-d-galactopyranosyl group from the substrate to form a covalent adduct to the enzyme; and (3) a larger 320 000-fold decrease in the first-order rate constant k(s) for hydrolysis of this covalent adduct. Only a small fraction (ca. 7%) of the 2-OH substituent effect is expressed in the ground-state Michaelis complex, so that the (apparent) strong interactions between the enzyme and 2-OH group that stabilize the transition state for beta-d-galactopyranosyl transfer only develop upon moving from the Michaelis complex to the transition state. Mg(2+) activates beta-galactosidase for cleavage of both 4-nitrophenyl-beta-d-galactopyranoside and 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside. This suggests that Mg(2+) activation does not involve interactions with the 2-OH group. The removal of Mg(2+) from beta-galactosidase causes a change in the rate-determining step for enzyme-catalyzed hydrolysis of 4-nitrophenyl-2-deoxy-beta-d-galactopyranoside from breakdown to formation of the covalent intermediate. The observed 2-OH effect would require a very large (10-11 kcal/mol) stabilization of the transition state for beta-d-galactopyranosyl group transfer to water by interactions between beta-galactosidase and the neutral 2-OH group. We suggest that the apparent effect of the neutral substituent is more simply rationalized by ionization of the 2-OH to form a 2-O(-) anion, which provides effective electrostatic stabilization of the cationic transition state for glycoside cleavage at an active site of relatively low dielectric constant.

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Section: Hydrolysis Of 2-deoxy-β-d-galactopyranosylated Enzymementioning

confidence: 80%

Ground-State, Transition-State, and Metal-Cation Effects of the 2-Hydroxyl Group on β-d-Galactopyranosyl Transfer Catalyzed by β-Galactosidase (Escherichia coli, lac Z)

et al. 2005

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“…Considering the charge repulsion between the Glu δ-carboxylate and the two neighboring side chain carboxylates of Asp7 and Glu′147, it is conceivable that the former group displays a strongly shifted pK a value that might enable a (partial) protonation at pH optimum, as observed, e.g., for β-galactosidase (20). Such a protonation can be expected to have little impact on the hydrogen bonding network between the three carboxylates and surrounding crystal waters (vide infra), nor is it expected to have an impact on the reaction chemistry.…”

Section: Resultsmentioning

confidence: 96%

Multiple Substrate Binding States and Chiral Recognition in Cofactor-independent Glutamate Racemase: A Molecular Dynamics Study

Möbitz

Bruice

2004

Biochemistry

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Glutamate racemase (MurI) catalyzes the racemization of glutamate; two cysteine residues serve as catalytic acid and base. On the basis of the crystal structure of MurI from the hyperthermophilic bacterium Aquifex pyrophilus, we performed molecular dynamics (MD) simulations of six different systems to investigate stereochemistry, substrate ligation, and active site protonation state. The catalytic competence of individual systems was assessed by the abundance of reactive conformers. Only systems in which Cys70 is poised to deprotonate d-Glu were found to be catalytically competent (idem Cys178/l-Glu), in agreement with the experimentally observed stereochemistry of Lactobacillus fermentii MurI [Tanner, M. E. et al. (1993) Biochemistry 32, 3998-4006]. Only systems in which the alpha-amino group of l/d-Glu and the imidazole moiety of His are deprotonated are catalytically competent. The active site of MurI displays an unusual flexibility in substrate ligation, and several transitions between stable binding patterns were observed. In catalytically competent binding states, the conserved threonine residues 72, 114, and 117 ligate the alpha-carboxylate of Glu and the Asn71 amides ligate the alpha-amino group of Glu, whereas the delta-carboxylate of Glu is steered by electrostatic repulsion from the Asp7 and Glu147 side chain carboxylates. A network of hydrogen bonds controls the positioning of each thiol/thiolate. In what we term substrate flipping, Glu suddenly rotates into a binding pattern that resembles the post-racemization state of the other enantiomer, i.e., each enantiomer can be bound in two distinct states. Substrate flipping and unfavorable substrate binding successively trigger dissociation of the substrate, accompanied by an opening of the active site channel. We explain how the weak binding of Glu contributes to catalysis and suggest a mechanism by which binding mismatches are propagated into an opening of the active site.

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“…The second proton 'in-flight' in the rate-limiting transition state for which these data provide evidence is likely the proton that is thought to be donated by the imidazolium of His 12 to the 2 0 -oxygen [50]. b-galactosidase proceeds through the intermediacy of a galactosyl-enzyme whose hydrolytic decomposition rate-limits k c for substrates with sufficiently reactive aglycone leaving groups where formation of this intermediate is rapid [54][55][56][57]. b-galactosidase proceeds through the intermediacy of a galactosyl-enzyme whose hydrolytic decomposition rate-limits k c for substrates with sufficiently reactive aglycone leaving groups where formation of this intermediate is rapid [54][55][56][57].…”

Section: Double-displacement Mechanisms -Secondmentioning

confidence: 85%

“…Some of the most compelling evidence for this comes from recent mutagenesis experiments in which the E461G enzyme was shown to have pH-rate dependences and solvent isotope effects predicted for this mutant [57]. Specifically for the latter property, the solvent isotope effect on k c for hydrolysis of 4-nitrophenyl b-D-galactopyranoside of 1.7 for wild-type enzyme [54] was reduced to a value near unity with the E461G mutant [57]. Since k c is thought to be rate-limited by galactosyl-enzyme formation, and it is known that nucleophilic attack on the anomeric carbon by Glu 537 is not subject to general-catalysis [55,56], the observed solvent isotope effect for the native enzyme must be due the establishment of a proton bridge between the protonated carboxyl moiety of Glu 461 and the oxygen of the leaving group.…”

Section: Single Displacement Mechanismsmentioning

confidence: 99%

Proton Transfer during Catalysis by Hydrolases

Stein

2006

Hydrogen‐Transfer Reactions

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

Cited by 3 publications

References 21 publications

Ground-State, Transition-State, and Metal-Cation Effects of the 2-Hydroxyl Group on β-d-Galactopyranosyl Transfer Catalyzed by β-Galactosidase (Escherichia coli, lac Z)

Ground-State, Transition-State, and Metal-Cation Effects of the 2-Hydroxyl Group on β-d-Galactopyranosyl Transfer Catalyzed by β-Galactosidase (Escherichia coli, lac Z)

Multiple Substrate Binding States and Chiral Recognition in Cofactor-independent Glutamate Racemase: A Molecular Dynamics Study

Proton Transfer during Catalysis by Hydrolases

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