@article {4780,
	title = {Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate},
	journal = {Protein Science},
	volume = {9},
	number = {3},
	year = {2000},
	note = {ISI Document Delivery No.: 295XWTimes Cited: 16Cited Reference Count: 56},
	month = {Mar},
	pages = {512-524},
	type = {Article},
	abstract = {NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. N-15 T-1, T-2, and N-15{H-1} NOE values were measured for similar to 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time ( tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S-2 = 0.86 +/- 0.04 and S-2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the,glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by N-15-NMR relaxation measurements.},
	keywords = {ACTIVE-SITE NUCLEOPHILE, ANISOTROPIC ROTATIONAL, BACKBONE DYNAMICS, BINDING, BURIED NEUTRAL HISTIDINE, DIFFUSION, glucanase, GLYCOSYL-ENZYME INTERMEDIATE, MAGNETIC-RESONANCE RELAXATION, MODEL-FREE APPROACH, N-15 NMR RELAXATION, NMR, order parameter, PROTEIN, PROTEINS, relaxation dynamics, SIDE-CHAIN RESONANCES, STRUCTURE},
	isbn = {0961-8368},
	url = {<Go to ISI>://000085994800009},
	author = {Connelly, G. P. and Withers, S. G. and McIntosh, L. P.}
}
@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.}
}
