@article {5098,
	title = {Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase},
	journal = {Biochemistry},
	volume = {40},
	number = {34},
	year = {2001},
	note = {ISI Document Delivery No.: 466CYTimes Cited: 56Cited Reference Count: 50},
	month = {Aug},
	pages = {10115-10139},
	type = {Article},
	abstract = {Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (GIu78, pK(a) 4.6) and the acid/base catalyst (Glu 172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pKa values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pKa values (measured directly by C-13 NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu 172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pKa calculations show that Glu78 has a lower pKa value than Glu 172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pKa value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pKa values of Glu78 and Glu 172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pKa values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pKa value is largely dependent on the length of the hydrogen bond (shorter bonds lower pKa values more) and the chemical nature of the donor (COOH > OH > CONH2). In contrast, neighboring carboxyl groups can either lower or raise the pKa values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.},
	keywords = {ASSIGNMENTS, BACILLUS-CIRCULANS XYLANASE, CATALYSIS, GLYCOSYL-ENZYME INTERMEDIATE, KINETIC-ANALYSIS, MASS-SPECTROMETRY, MECHANISMS, NMR, PK(A), PROTEIN, SITE NUCLEOPHILE, structures},
	isbn = {0006-2960},
	url = {<Go to ISI>://000170627800014},
	author = {Joshi, M. D. and Sidhu, G. and Nielsen, J. E. and Brayer, G. D. and Withers, S. G. and McIntosh, L. P.}
}
@article {4842,
	title = {Hydrogen bonding and catalysis: A novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase},
	journal = {Journal of Molecular Biology},
	volume = {299},
	number = {1},
	year = {2000},
	note = {ISI Document Delivery No.: 318NCTimes Cited: 96Cited Reference Count: 58},
	month = {May},
	pages = {255-279},
	type = {Article},
	abstract = {The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pK(a) 4.6) and the acid/base catalyst Glu172 (pK(a) 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an similar to 20 \% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pK(a) values of 3.5 and 5.8. Based on C-13-NMR titrations, the predominant pK(a) values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pK(a), is protonated, while Glu78, with the higher pK(a), is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only similar to 1\% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 Angstrom) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis. (C) 2000 Academic Press.},
	keywords = {3-DIMENSIONAL STRUCTURE, 4-BETA-XYLANASES, ACTIVE-SITE, BACILLUS-CIRCULANS XYLANASE, CYCLODEXTRIN GLYCOSYLTRANSFERASE, electrostatics, ENDO-1, GLYCOSYL-ENZYME INTERMEDIATE, ISOTOPE, MOLECULAR-WEIGHT, NMR, NUCLEAR-MAGNETIC-RESONANCE, nucleophile, PANCREATIC ALPHA-AMYLASE, pH-dependent enzyme mechanism, SHIFT, TRICHODERMA-REESEI, X-RAY CRYSTALLOGRAPHY, X-ray structure},
	isbn = {0022-2836},
	url = {<Go to ISI>://000087289400019},
	author = {Joshi, M. D. and Sidhu, G. and Pot, I. and Brayer, G. D. and Withers, S. G. and McIntosh, L. P.}
}
@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 {4197,
	title = {Mechanisms of cellulases and xylanases},
	journal = {Biochemical Society Transactions},
	volume = {26},
	number = {2},
	year = {1998},
	note = {ISI Document Delivery No.: ZT170Times Cited: 22Cited Reference Count: 24664th Meeting of the Biochemical-SocietyDEC 15-17, 1997READING, ENGLANDBiochem Soc},
	month = {May},
	pages = {156-160},
	type = {Proceedings Paper},
	keywords = {ACID-SEQUENCE SIMILARITIES, ACTIVE-SITE, BACILLUS-CIRCULANS XYLANASE, CLASSIFICATION, ENZYME, FAMILIES, GLUCOSIDASE, glycosidase, GLYCOSYL HYDROLASES, HYDROLYSIS, nucleophile},
	isbn = {0300-5127},
	url = {<Go to ISI>://000074056400019},
	author = {Birsan, C. and Johnson, P. and Joshi, M. and MacLeod, A. and McIntosh, L. and Monem, V. and Nitz, M. and Rose, D. R. and Tull, D. and Wakarchuck, W. W. and Wang, Q. and Warren, R. A. J. and White, A. and Withers, S. G.}
}
@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.}
}
