@article {4667,
	title = {Sugar ring distortion in the glycosyl-enzyme intermediate of a family G/11 xylanase},
	journal = {Biochemistry},
	volume = {38},
	number = {17},
	year = {1999},
	note = {ISI Document Delivery No.: 193YMTimes Cited: 122Cited Reference Count: 50},
	month = {Apr},
	pages = {5346-5354},
	type = {Article},
	abstract = {The 1.8 Angstrom resolution structure of the glycosyl-enzyme intermediate formed on the retaining beta-1,4-xylanase from Bacillus circulans has been determined using X-ray crystallographic techniques. The 2-fluoro-xylose residue bound in the -1 subsite adopts a B-2,B-5 (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism. Comparison of this structure to that of a mutant of this same enzyme noncovalently complexed with xylotetraose [Wakarchuk et al. (1994) Protein Sci. 3, 467-475] reveals a number of differences beyond the distortion of the sugar moiety. Most notably, a bifurcated hydrogen bond interaction is formed in the glycosyl-enzyme intermediate involving H-eta of Tyr69, the endocyclic oxygen (O5) of the xylose residue in the -1 subsite, and O-epsilon 2 of the catalytic nucleophile, Glu78. To gain additional understanding of the role of Tyr69 at the active site of this enzyme, we also determined the 1.5 Angstrom resolution structure of the catalytically inactive Tyr69Phe mutant. Interestingly, no significant structural perturbation due to the loss of the phenolic group is observed. These results suggest that the interactions involving the phenolic group of Tyr69, O5 of the proximal saccharide, and Glu78 O-epsilon 2 important for the catalytic mechanism of this enzyme, and it is proposed that, through charge redistribution, these interactions serve to stabilize the oxocarbenium-like ion of the transition state. Studies of the covalent glycosyl-enzyme intermediate of this xylanase also provide insight into specificity, as contacts with C5 of the xylose moiety exclude sugars with hydroxymethyl substituents, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis.},
	keywords = {ACTIVE-SITE RESIDUES, BACILLUS-CIRCULANS XYLANASE, beta-glucosidase, CELLULOMONAS-FIMI, glycosidase, IDENTIFICATION, LEAVING GROUP, MECHANISM, REACTION COORDINATE, TRICHODERMA-REESEI},
	isbn = {0006-2960},
	url = {<Go to ISI>://000080165400009},
	author = {Sidhu, G. and Withers, S. G. and Nguyen, N. T. and McIntosh, L. P. and Ziser, L. and Brayer, G. D.}
}
@article {4447,
	title = {Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry},
	journal = {Biochemistry},
	volume = {37},
	number = {21},
	year = {1998},
	note = {ISI Document Delivery No.: ZR717Times Cited: 73Cited Reference Count: 53},
	month = {May},
	pages = {7664-7669},
	type = {Article},
	abstract = {For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-D-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2,5-dinitrophenol with stopped-flow W-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.},
	keywords = {ACTIVE-SITE NUCLEOPHILE, AMINO-ACID, BACILLUS-CIRCULANS XYLANASE, beta-glucosidase, CELLULASES, CYTOCHROME-C, GAS-PHASE, IDENTIFICATION, inhibitors, MECHANISMS},
	isbn = {0006-2960},
	url = {<Go to ISI>://000074006700002},
	author = {Zechel, D. L. and Konermann, L. and Withers, S. G. and Douglas, D. J.}
}
@article {4069,
	title = {Design and synthesis of 2{\textquoteright}-deoxy-2{\textquoteright}-fluorodisaccharides as mechanism-based glycosidase inhibitors that exploit aglycon specificity},
	journal = {Journal of the American Chemical Society},
	volume = {119},
	number = {25},
	year = {1997},
	note = {ISI Document Delivery No.: XG882Times Cited: 37Cited Reference Count: 23},
	month = {Jun},
	pages = {5792-5797},
	type = {Article},
	abstract = {Stable, aglycon-specific inactivators of glycosidases have considerable potential as fools in the study of mechanisms of oligosaccharide processing, and possibly as avenues toward new therapeutics. Glycosidases for which the rate-determining step with the natural substrate is the hydrolysis of the glycosyl-enzyme intermediate are shown to be inactivated by the 2{\textquoteright}-deoxy-2{\textquoteright}-fluoro derivative of this substrate. Thus Agrobacterium faecalis beta-glucosidase is inactivated by 2{\textquoteright}-deoxy-2{\textquoteright}-fluorocellobiose according to inactivation parameters of k(i) = 0.018 min(-1) and K-i = 20 mM. Inactivation is shown to occur via the accumulation of the same 2-deoxy-2-fluoroglycosyl-enzyme intermediate as that formed using activated 2-deoxy-2-fluoroglycosides by identification of the labeled peptide in proteolytic digests. Thus, interactions between the enzyme and the sugar aglycon provide sufficient transition state stabilization to allow formation and trapping of the glycosyl-enzyme. beta-Glucocerebrosidase, a beta-glucosidase specific for hydrolysis of glucocerebrosides, is not inactivated by 2{\textquoteright}-deoxy-2{\textquoteright}-fluorocellobiose, thereby demonstrating the aglycon specificity of this class of inactivator.},
	keywords = {ACTIVE-SITE NUCLEOPHILE, beta-glucosidase, GALACTOSIDASE, IDENTIFICATION, residue, TANDEM MASS-SPECTROMETRY},
	isbn = {0002-7863},
	url = {<Go to ISI>://A1997XG88200003},
	author = {McCarter, J. D. and Yeung, W. and Chow, J. and Dolphin, D. and Withers, S. G.}
}
@article {3762,
	title = {The pK(a) of the general acid/base carboxyl group of a glycosidase cycles during catalysis: A C-13-NMR study of Bacillus circuluns xylanase},
	journal = {Biochemistry},
	volume = {35},
	number = {31},
	year = {1996},
	note = {ISI Document Delivery No.: VB211Times Cited: 149Cited Reference Count: 43},
	month = {Aug},
	pages = {9958-9966},
	type = {Article},
	abstract = {The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water, The dual role of Glu172 places specific demands upon its ionization states and hence pK(a) values. C-13-NMR titrations of xylanase, labeled with [delta-C-13]glutamic acid, have revealed pK(a) values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pK(a) values obtained from a study of the pH dependence of k(cat)/K-m and demonstrate that, at the enzyme{\textquoteright}s pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pK(a) for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2{\textquoteright},4{\textquoteright}-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032], A similar pK(a) is measured for Glu172 when a glutamine is present at position 78. This large decrease in pK(a) of similar to 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such {\textquoteright}{\textquoteright}pK(a) cycling{\textquoteright}{\textquoteright} during catalysis is likely to be a common phenomenon in glycosidases.},
	keywords = {ACTIVE-SITE RESIDUES, AMINO-ACIDS, beta-glucosidase, ELECTROSTATIC INTERACTIONS, ESCHERICHIA-COLI, IDENTIFICATION, LYSOZYME, MECHANISMS, NUCLEAR-MAGNETIC-RESONANCE, PROTEINS},
	isbn = {0006-2960},
	url = {<Go to ISI>://A1996VB21100003},
	author = {McIntosh, L. P. and Hand, G. and Johnson, P. E. and Joshi, M. D. and Korner, M. and Plesniak, L. A. and Ziser, L. and Wakarchuk, W. W. and Withers, S. G.}
}
@article {2871,
	title = {SYNTHESES OF 2-DEOXY-2-FLUORO MONOSACCHARIDE AND OLIGOSACCHARIDE GLYCOSIDES FROM GLYCALS AND EVALUATION AS GLYCOSIDASE INHIBITORS},
	journal = {Carbohydrate Research},
	volume = {249},
	number = {1},
	year = {1993},
	note = {ISI Document Delivery No.: MD387Times Cited: 41Cited Reference Count: 34},
	month = {Oct},
	pages = {77-90},
	type = {Article},
	abstract = {Several fluorinated oligosaccharides, including 2-deoxy-2-fluoro derivatives of cellobiose, maltose, and maltotriose were synthesized by the action of fluorine or acetyl hypofluorite on the corresponding glycal peracetates. Temperature effects on the stereoselectivities of these reactions were examined. Addition of acetyl hypofluorite to several 2-substituted glycals in the gluco or galacto series gave 2,2-disubstituted arabino- or lyxo-hexose derivatives; 3,4,6-tri-O-acetyl-2-fluoro-D-glucal or the analogous galactal yielded 2-deoxy-2,2-difluoro arabino- or lyxo-hexose peracetates, whereas 2-acetoxy-3,4,6-tri-O-acetyl-D-glucal or the analogous galactal gave 2(R)-2-acetoxy-2-fluoro-arabino- or lyxo-hexose peracetates, respectively. 2-Acetamido-3,4,6-tri-O-acetyl-D-glucal gave 2(R)-2-acetamido-2-acetoxy-3,4,6-tri-O-acetyl-alpha-D-arabino-hexopyrano syl fluoride. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside was an inactivator of the exoglucanase from Cellulomonas fimi while 2-deoxy-2-fluoro-alpha-maltosyl and alpha-maltotriosyl fluorides were slow substrates of human pancreatic alpha-amylase and rabbit muscle glycogen debranching enzyme, respectively.},
	keywords = {ACETYL HYPOFLUORITE, beta-glucosidase, CATALYTIC FLEXIBILITY, DERIVATIVES, ENZYME, GLYCOSYLASES, hydration, INTERMEDIATE, MECHANISM},
	isbn = {0008-6215},
	url = {<Go to ISI>://A1993MD38700007},
	author = {McCarter, J. D. and Adam,Michael J. and Braun, C. and Namchuk, M. and Tull, D. and Withers, S. G.}
}
