Solvent Deuterium Isotope Effect on the Binding of β-d-Galactopyranosyl Derivatives to β-Galactosidase (Escherichia coli, lac Z)

Richard, John P.; McCall, Deborah A.

doi:10.1006/bioo.1999.1157

Cited by 8 publications

(9 citation statements)

References 17 publications

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“…The nature of the transition states for both steps has also been probed using isotope effects (16,19,20,22). These experiments suggest that the transition state for step 2 is more planar (sp 2 hybridized) than the starting state (the intermediate), which is consistent with an oxocarbenium ion transition state, as has been proposed for many other glycosidases.…”

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confidence: 55%

“…Structure/reactivity relationships suggest that there is at least partial acid assistance of leaving group departure, especially with more basic leaving groups such as glucose (16)(17)(18). A magnesium ion appears to be necessary for such acid assistance, although the details are somewhat controversial (12,17,(19)(20)(21)(22). All of the kinetic data are consistent with either Lewis catalysis by the magnesium or Bronsted catalysis by Glu461.…”

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confidence: 83%

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A Structural View of the Action ofEscherichia coli(lacZ) β-Galactosidase^,

et al. 2001

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The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.

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confidence: 55%

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confidence: 83%

A Structural View of the Action ofEscherichia coli(lacZ) β-Galactosidase^,

et al. 2001

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“…This, of course, could be due to a fortuitous cancellation of isotope effects on k cat and K M as have been seen with other glycohydrolases (e.g., [18]). Indeed, at the pL optima, the hydrolysis of GlcSBiz to the enzyme does show a very small, but significant, solvent isotope effect on k cat [=1.28(±0.06)].…”

Section: Discussionmentioning

confidence: 85%

“…For this substrate, the rate-limiting step is most likely glucosylation of the enzyme 5 and the SKIE indicates proton transfer in, or before, this step. This is not the case with other substrates such as pNPG [17], phenyl β-glucoside [18], methyl β-glucoside [19] or the thioglucoside, pNPSG [6], all of which show no solvent kinetic isotope effect ( D 2 O k cat ≈1).…”

Section: Discussionmentioning

confidence: 99%

Reactive thioglucoside substrates for β-glucosidase

Avegno¹,

Hasty²,

Parameswar³

et al. 2013

Archives of Biochemistry and Biophysics

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A new, very efficient, class of thioglycoside substrates has been found for β-glucosidase. While thioglycosides are usually resistant to hydrolysis, even in the presence of acids or most glycohydrolases, the β-D-glucopyranosides of 2- mercaptobenzimidazole (GlcSBiz) and 2-mercaptobenzoxazole (GlcSBox) have been found to be excellent substrates for β-glucosidase from both sweet almond (a family 1 glycohydrolase) and Aspergillus niger (a family 3 glycohydrolase), reacting nearly as well as p-nitrophenyl β-D-glucoside. The enzyme-catalyzed hydrolysis of GlcSBiz proceeds with retention of configuration. As with the (1000-fold slower) hydrolysis of phenyl thioglucosides catalyzed by the almond enzyme, the pL (pH/pD)-independent kcat/KM does not show a detectable solvent deuterium kinetic isotope effect (SKIE), but unlike the hydrolysis of phenyl thioglucosides, a modest SKIE is seen on kcat [D2Okcat = 1.28 (±0.06)] at the pL optimum (5.5 ≤ pL ≤ 6.6). A solvent isotope effect is also seen on the KM for the N-methyl analog of GlcSBiz. These results suggest that the mechanism for the hydrolysis of the β-thioglucoside of 2-mercaptobenzimidazole and of 2- mercaptobenzoxazole involves remote site protonation (at the ring nitrogen) followed by cleavage of the thioglucosidic bond resulting in the thione product.

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“…The difference in the SDIE on k cat /K m and k cat for the wild-type enzyme might reflect either the isotope effect on the dissociation constant K d for substrate release (18) or a mechanism where substrate binding is partly rate-determining for k cat /K m , but not for k cat (19). The change to a smaller difference between the SDIE on k cat /K m and k cat for the E461G ␤-galactosidase might then represent an effect of this mutation on the SDIE for the dissociation constant K d ; however, we are not aware of any precedent for such an effect of a point mutation of the SDIE for substrate binding.…”

Section: Discussionmentioning

confidence: 99%