@article {2461,
	title = {Direct Measurement of the Kinetics of CBM9 Fusion-Tag Bioprocessing Using Luminescence Resonance Energy Transfer},
	journal = {Biotechnology Progress},
	volume = {25},
	number = {3},
	year = {2009},
	note = {ISI Document Delivery No.: 462SETimes Cited: 1Cited Reference Count: 44Kavoosi, Mojgan Creagh, A. Louise Turner, Robin F. B. Kilburn, Douglas G. Haynes, Charles A.},
	month = {May-Jun},
	pages = {874-881},
	type = {Article},
	abstract = {The economics of affinity-tagging technologies, particularly at preparative scales, depends in part on the cost and efficiency of the bioprocessing step used to remove the affinity tag and obtain the final purified product (Lowe et al., J Biochem Biophys Methods. 2001,49:561-574). When CBM9, the family 9 cellulose binding module from Thermotoga maritima, serves as the affinity tag, the overall efficiency of tag removal is a function of the choice of processing enzyme and the local structure of the cleavage site, most notably the linker sequence flanking the bioprocessing recognition site on the tag side. A novel spectroscopic method is reported and used to rapidly and accurately measure CBM9 fusion-tag bioprocessing kinetics and their dependence on the choice of linker sequence. The assay monitors energy transfer between a lanthanide-based donor bound to the CBM9 tag and an acceptor fluorophore presented on the tat-get protein or peptide. Enzyme-catalyzed cleavage of the fusion tag terminates this resonance energy transfer, resulting in a change in fluorescence intensity that can be monitored to quantify substrate concentration over time. The assay is simple, fast and accurate, providing k(cat)/K-M values that contain standard errors of less than 3\%. As a result, both substantial and subtle differences in bioprocessing kinetics can be measured and used to guide bioproduct design. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 874-881, 2009},
	keywords = {10A, ACTIVE-SITE, affinity chromatography, BEND ANGLE, CARBOHYDRATE-BINDING MODULE, CONFORMATIONAL-CHANGES, enterokinase, ESCHERICHIA-COLI, fusion tag, green fluorescent protein (GFP), high throughput assay, LANTHANIDE, library, linker, luminescence, protein purification, PURIFICATION, RECOMBINANT PROTEINS, resonance energy transfer (LRET), RNA-POLYMERASE, terbium, THERMOTOGA-MARITIMA, XYLANASE},
	isbn = {8756-7938},
	url = {<Go to ISI>://000267375500030},
	author = {Kavoosi, M. and Creagh, A. L. and Turner, R. F. B. and Kilburn, D. G. and Haynes, C. A.}
}
@article {2635,
	title = {Probing General Acid Catalysis in the Hammerhead Ribozyme},
	journal = {Journal of the American Chemical Society},
	volume = {131},
	number = {3},
	year = {2009},
	note = {ISI Document Delivery No.: 427PQTimes Cited: 5Cited Reference Count: 58Thomas, Jason M. Perrin, David M.},
	month = {Jan},
	pages = {1135-1143},
	type = {Article},
	abstract = {Recent crystallographic and computational studies have provided fresh insights into the catalytic mechanism of the RNA-cleaving hammerhead ribozyme. Based on these findings, specific ribozyme functional groups have been hypothesized to act directly as the general acid and base catalysts, although the catalytic role of divalent metal cations (M2+) remains uncertain: We now report a functional characterization of the general acid catalysis mechanism and the role of an M2+ cofactor therein, for the S. mansoni hammerhead (an "extended" hammerhead ribozyme). We have compared hammerhead cleavage of substrates with natural (ribo-phosphodiester) versus bridging-5{\textquoteright}-phosphorothioate scissile linkages, in the contexts of active site mutations and M2+ substitution. Cleavage of the natural substrate is inhibited by modification of the G8 2{\textquoteright}-OH ribozyme residue and depends strongly upon the presence and identity of an M2+ cofactor; in contrast, cleavage of the bridging-phosphorothioate substrate is conspicuously insensitive to any of these factors. These results imply that (1) both an M2+ cofactor and the G8 2{\textquoteright}-OH play crucial roles in hammerhead general acid catalysis and (2) the M2+ cofactor does not contribute to general acid catalysis via Lewis acid stabilization of the leaving group. General acid pK(a) perturbation was also demonstrated for both M2+ substitution and G8 2{\textquoteright}-OH modification, which suggests transition state M2+ coordination of the G8 2{\textquoteright}-OH, to lower its pK(a) and improve its ability to transfer a proton to the leaving group. We also report a simple method for synthesizing radiolabeled bridging-5{\textquoteright}-phosphorothioate substrates.},
	keywords = {2{\textquoteright}-HYDROXYL GROUP, ACTIVE-SITE, BASE CATALYSIS, CATIONS, MEDIATED CLEAVAGE, METAL-ION, MONOVALENT, P-31 NMR, PHOSPHOLIPASE-C, RIBONUCLEASE-A, SUBSTRATE-ANALOG},
	isbn = {0002-7863},
	url = {<Go to ISI>://000264791600054},
	author = {Thomas, J. M. and Perrin,David M.}
}
@article {2285,
	title = {Probing General Base Catalysis in the Hammerhead Ribozyme},
	journal = {Journal of the American Chemical Society},
	volume = {130},
	number = {46},
	year = {2008},
	note = {ISI Document Delivery No.: 406QZTimes Cited: 8Cited Reference Count: 53Thomas, Jason M. Perrin, David M.},
	month = {Nov},
	pages = {15467-15475},
	type = {Article},
	abstract = {Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2{\textquoteright}-bromoacetamide group in place of the nucleophilic 2{\textquoteright}-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2{\textquoteright}-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg2+] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pK(a) of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.},
	keywords = {ACTIVE-SITE, CLEAVAGE, CRYSTAL-STRUCTURE, DELTA VIRUS RIBOZYME, HAIRPIN RIBOZYME, MECHANISM, METAL-ION, MONOVALENT CATIONS, NUCLEOBASE CATALYSIS, RNA},
	isbn = {0002-7863},
	url = {<Go to ISI>://000263311300051},
	author = {Thomas, J. M. and Perrin,David M.}
}
@article {2279,
	title = {Transient oxidation as a mechanistic strategy in enzymatic catalysis},
	journal = {Current Opinion in Chemical Biology},
	volume = {12},
	number = {5},
	year = {2008},
	note = {ISI Document Delivery No.: 375SZTimes Cited: 1Cited Reference Count: 41Tanner, Martin E.},
	month = {Oct},
	pages = {532-538},
	type = {Review},
	abstract = {Enzymes that employ a transient oxidation mechanism catalyze transformations that are overall redox neutral, but involve intermediates that have a higher oxidation state than the substrates or products. An oxidation/reduction sequence may be used directly to promote isomerization reactions or indirectly to permit the formation of stabilized intermediates such as carbanions. This review will focus on three recent examples of nicotinamide-dependent enzymes that have been found to employ transient oxidation during catalysis: ADP-L-glycero-D-manno-heptose 6-epimerase, GDP-mannose 3,5-epimerase, and the 6-phosphoglucosidases from family 4. These enzymes are remarkable in their ability to catalyze either nonstereospecific hydride transfers or multiple chemical steps within a single active site.},
	keywords = {ACTIVE-SITE, ALPHA-GLUCOSIDASE, BACILLUS-SUBTILIS, CRYSTAL-STRUCTURE, D-MANNO-HEPTOSE, D-MANNOHEPTOSE 6-EPIMERASE, DEHYDROQUINATE SYNTHASE, ESCHERICHIA-COLI, THERMOTOGA-MARITIMA, UDP-GALACTOSE 4-EPIMERASE},
	isbn = {1367-5931},
	url = {<Go to ISI>://000261134900010},
	author = {Tanner, M. E.}
}
@article {1135,
	title = {Spectroscopic studies of the anaerobic enzyme - Substrate complex of catechol 1,2-dioxygenase},
	journal = {Journal of the American Chemical Society},
	volume = {127},
	number = {48},
	year = {2005},
	note = {ISI Document Delivery No.: 990QVTimes Cited: 17Cited Reference Count: 86},
	month = {Dec},
	pages = {16882-16891},
	type = {Article},
	abstract = {The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/ substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C120), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.},
	keywords = {1, 2, 2-DIOXYGENASE, 3-DIHYDROXYBIPHENYL, 4-DIOXYGENASE, ACTIVE-SITE, CRYSTAL-STRUCTURE, DENSITY-FUNCTIONAL THEORY, EXTRADIOL DIOXYGENASES, IRON-TRANSPORT COMPOUNDS, ISOTOPIC-SUBSTITUTION SHIFTS, PROTOCATECHUATE 3, PSEUDOMONAS-ARVILLA C-1, RESONANCE RAMAN-SPECTROSCOPY},
	isbn = {0002-7863},
	url = {<Go to ISI>://000233759200038},
	author = {Horsman, G. P. and Jirasek, A. and Vaillancourt, F. H. and Barbosa, C. J. and Jarzecki, A. A. and Xu, C. L. and Mekmouche, Y. and Spiro, T. G. and Lipscomb, J. D. and Blades, M. W. and Turner, R. F. B. and Eltis, L. D.}
}
@article {1135,
	title = {Spectroscopic studies of the anaerobic enzyme - Substrate complex of catechol 1,2-dioxygenase},
	journal = {Journal of the American Chemical Society},
	volume = {127},
	number = {48},
	year = {2005},
	note = {ISI Document Delivery No.: 990QVTimes Cited: 17Cited Reference Count: 86},
	month = {Dec},
	pages = {16882-16891},
	type = {Article},
	abstract = {The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/ substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C120), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.},
	keywords = {1, 2, 2-DIOXYGENASE, 3-DIHYDROXYBIPHENYL, 4-DIOXYGENASE, ACTIVE-SITE, CRYSTAL-STRUCTURE, DENSITY-FUNCTIONAL THEORY, EXTRADIOL DIOXYGENASES, IRON-TRANSPORT COMPOUNDS, ISOTOPIC-SUBSTITUTION SHIFTS, PROTOCATECHUATE 3, PSEUDOMONAS-ARVILLA C-1, RESONANCE RAMAN-SPECTROSCOPY},
	isbn = {0002-7863},
	url = {<Go to ISI>://000233759200038},
	author = {Horsman, G. P. and Jirasek, A. and Vaillancourt, F. H. and Barbosa, C. J. and Jarzecki, A. A. and Xu, C. L. and Mekmouche, Y. and Spiro, T. G. and Lipscomb, J. D. and Blades, M. W. and Turner, R. F. B. and Eltis, L. D.}
}
@article {454,
	title = {Dehydroalanine-based inhibition of a peptide epimerase from spider venom},
	journal = {Journal of Organic Chemistry},
	volume = {67},
	number = {24},
	year = {2002},
	note = {ISI Document Delivery No.: 620AATimes Cited: 17Cited Reference Count: 42},
	month = {Nov},
	pages = {8389-8394},
	type = {Article},
	abstract = {Ribosomally produced peptides that contain D-amino acids have been isolated from a number of vertebrate and invertebrate sources. In each case, the D-amino acids are introduced by a posttranslational modification of a parent peptide containing only amino acids of the L-configuration. The only known enzyme to catalyze such a reaction is the peptide epimerase (also known as peptide isomerase) from the venom of the funnel web spider, Agelenopsis aperta. This enzyme interconverts two 48-amino-acid-long peptide toxins that differ only by the stereochemistry at a single serine residue. In this paper we report the synthesis and testing of two pentapeptide analogues that contain modified amino acids at the site normally occupied by the substrate serine residue. When the L-chloroalanine-containing peptide 3 was incubated with the epimerase it was converted into the dehydroalanine-containing peptide 4 via an elimination of HCl. The dehydroalanine peptide 4 was independently synthesized and found to act as a potent inhibitor of the epimerase (IC50 = 0.5 muM). These results support a direct deprotonation/reprotonation mechanism in which a carbanionic intermediate is formed. The observed inhibition by 4 can be attributed to the sp(2)-hybridization of the a-carbon in the dehydroalanine unit that mimics the planar geometry of the anionic intermediate.},
	keywords = {ACID, ACTIVE-SITE, ENZYME, GLUTAMATE RACEMASE, GRAMICIDIN-S SYNTHETASE, INFLUENZAE DIAMINOPIMELATE EPIMERASE, INITIATION MODULE PHEATE, MECHANISM, PROLINE RACEMASE, RESIDUES},
	isbn = {0022-3263},
	url = {<Go to ISI>://000179509400011},
	author = {Murkin, A. S. and Tanner, M. E.}
}
@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 {4553,
	title = {The inactivation of histidine ammonia-lyase by L-cysteine and oxygen: Modification of the electrophilic center},
	journal = {Journal of the American Chemical Society},
	volume = {121},
	number = {46},
	year = {1999},
	note = {ISI Document Delivery No.: 261NBTimes Cited: 10Cited Reference Count: 23},
	month = {Nov},
	pages = {10840-10841},
	type = {Article},
	keywords = {ACTIVE-SITE, DEHYDROALANINE, ENZYME, PRECURSOR, PSEUDOMONAS-PUTIDA, SERINE-143},
	isbn = {0002-7863},
	url = {<Go to ISI>://000084014300026},
	author = {Galpin, J. D. and Ellis, B. E. and Tanner, M. E.}
}
@article {4406,
	title = {Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains},
	journal = {Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire},
	volume = {76},
	number = {2-3},
	year = {1998},
	note = {ISI Document Delivery No.: 157QRTimes Cited: 11Cited Reference Count: 34},
	pages = {379-390},
	type = {Article},
	abstract = {The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the H-1,C-13, and N-15 signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and C-13-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.},
	keywords = {ACTIVE-SITE, ANGLE, aromatic residue, BACKBONE H-1, C-13-LABELED PROTEINS, CHEMICAL-SHIFTS, dihedral, DNA-BINDING, HETERONUCLEAR NMR, HISTIDINE-RESIDUES, MAGNETIC-RESONANCE SPECTROSCOPY, NMR assignment, NOE, pH titration, PHENYLALANINE RESIDUES, TRANSCRIPTION FACTOR},
	isbn = {0829-8211},
	url = {<Go to ISI>://000078073600026},
	author = {Slupsky, C. M. and Gentile, L. N. and McIntosh, L. P.}
}
@article {4235,
	title = {Characterization of a buried neutral histidine in Bacillus circulans xylanase: Internal dynamics and interaction with a bound water molecule},
	journal = {Biochemistry},
	volume = {37},
	number = {7},
	year = {1998},
	note = {ISI Document Delivery No.: YZ886Times Cited: 13Cited Reference Count: 59},
	month = {Feb},
	pages = {1810-1818},
	type = {Article},
	abstract = {NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule. Rate constants for the specific acid-and base-catalyzed exchange following bimolecular kinetics (EX2) of the nitrogen-bonded H-epsilon 2 of this buried, neutral histidine were determined, At pD(min) 7.0 and 30 degrees C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of similar to 10(7) relative to that predicted for an exposed histidine, The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations. Consistent with its buried environment, the N-epsilon 2-H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S-2 = 0.83 determined from N-15 relaxation measurements, The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H-epsilon 2 of His149. Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H-epsilon 2 and a water molecule with the same chemical shift as the bulk solvent. However, the chemical shift of H-epsilon 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 +/- 0.02) indicate that this is nor a so-called low-barrier hydrogen bond. Lower and upper bounds on the lifetime of the internal water are estimated to be 10(-8) and 10(-3) s. Therefore the chemical exchange of solvent protons with those of His149 H-epsilon 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude.},
	keywords = {ACTIVE-SITE, AMIDE PROTON-EXCHANGE, ASSIGNMENTS, DOMAIN, HYDROGEN-BOND, NMR-SPECTROSCOPY, NUCLEAR MAGNETIC-RESONANCE, PROTEINS, relaxation, TRICHODERMA-REESEI},
	isbn = {0006-2960},
	url = {<Go to ISI>://000072304200010},
	author = {Connelly, G. P. and McIntosh, L. P.}
}
@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 {3799,
	title = {Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange},
	journal = {Protein Science},
	volume = {5},
	number = {11},
	year = {1996},
	note = {ISI Document Delivery No.: VQ786Times Cited: 41Cited Reference Count: 63},
	month = {Nov},
	pages = {2319-2328},
	type = {Article},
	abstract = {Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the H-1, C-13, and N-15 resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded H-1(epsilon 2) of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 X 10(-5) s(-1) at pH* 7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pK(a) of His 156 is unperturbed at similar to 6.5, as measured from the pH dependence of the N-15- and H-1-NMR spectra of BCX. In contrast, His 149 has a pK(a) < 2.3, existing in the neutral (NH)-H-epsilon 2 tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.},
	keywords = {ACTIVE-SITE, ALPHA-HELICES, AQUEOUS-SOLUTION, CHEMICAL-SHIFTS, ESCHERICHIA-COLI, GLYCOSYL HYDROLASES, H-1-NMR SPECTRUM, HYDROGEN-DEUTERIUM EXCHANGE, IMIDAZOLE, internal water, L-ALA-OH, NUCLEAR MAGNETIC-RESONANCE, pH titration, pK(alpha), PROTEIN HYDRATION, protein stability},
	isbn = {0961-8368},
	url = {<Go to ISI>://A1996VQ78600018},
	author = {Plesniak, L. A. and Connelly, G. P. and Wakarchuk, W. W. and McIntosh, L. P.}
}
@article {3621,
	title = {Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification},
	journal = {Protein Science},
	volume = {5},
	number = {11},
	year = {1996},
	note = {ISI Document Delivery No.: VQ786Times Cited: 41Cited Reference Count: 34},
	month = {Nov},
	pages = {2311-2318},
	type = {Article},
	abstract = {The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the p-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70\% and 90\%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.},
	keywords = {absorbance spectroscopy, ACTIVE-SITE, CELLOBIOHYDROLASE-I, cellulose-binding, ENDOGLUCANASE, ESCHERICHIA-COLI, FIMI, fluorescence spectroscopy, N-bromosuccinimide, NMR, NUCLEAR-MAGNETIC-RESONANCE, SCHIZOPHYLLUM-COMMUNE, SPECTROSCOPY, TRICHODERMA-REESEI, XYLANASE-A},
	isbn = {0961-8368},
	url = {<Go to ISI>://A1996VQ78600017},
	author = {Bray, M. R. and Johnson, P. E. and Gilkes, N. R. and McIntosh, L. P. and Kilburn, D. G. and Warren, R. A. J.}
}
