Effects of both Shortening and Lengthening the Active Site Nucleophile of<i> Bacillus circulans</i> Xylanase on Catalytic Activity

Lawson, Sherry L.; Wakarchuk, Warren W.; Withers, Stephen G.

doi:10.1021/bi960586v

Cited by 57 publications

(69 citation statements)

References 29 publications

(41 reference statements)

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“…Notably, the active-site peptide of Bcx was not isolated from model proteome B (Fig. 3B), which contained mutant E78C Bcx (mutated catalytic nucleophile), in agreement with the previous finding that E78C Bcx is not capable of catalyzing the glycosylation reaction, the first step in the double-displacement mechanism (35). Model proteome C demonstrated the concurrent isolation of the active-site peptides of both target enzymes (Fig.…”

Section: Detection Of the Affinity-isolated Labeled Active-site Peptsupporting

confidence: 90%

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Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining ␤-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining ␤-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi ␤-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank TM accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ϳ70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.With the completion of the genome sequences of many organisms, the field of proteomics faces several major tasks. One challenge is that of identification and assignment of structure/function to the tens of thousands of proteins encoded by prokaryotic and eukaryotic genomes. Another challenge is accurate quantitative analysis of changes in protein levels/activities that occur within a proteome as a response to biological perturbations that are due either to normal developmental and metabolic changes or to abnormalities associated with disease. Proteomic techniques such as comparative two-dimensional gel electrophoresis coupled with mass spectrometry, the isotope-coded affinity tagging approach (1), and variations of isotope-coded affinity tagging that identify sites of modification (e.g. glycosylation and phosphorylation) on proteins (2-4) fail to provide a direct assessment of protein function.Recently, several chemical strategies for activity-based protein profiling (ABPP) 4 in complex proteomes that target several enzyme groups, including oxidoreductases (5, 6), serine hydrolases (7, 8), cysteine proteases (9, 10), threonine proteases (11), metalloproteases (12), protein phosphatases (13), kinases (14), and exoglycosidases (15), have been employed. ABPP probes have two general features: 1) an active sitedirected (mechanism-based) inactivator or affinity label that reacts with a catalytic residue and forms a covalent adduct with the target enzyme(s) and 2) one or more reporter groups that enable rapid detection (e.g. a fluorophore) and/or affinity isolation (e.g. biotin) (16). As such, ABPP methods can provide direct information on post-translational forms of protein regulation (17). However, most of the ABPP research ...

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Section: Detection Of the Affinity-isolated Labeled Active-site Peptsupporting

confidence: 90%

“…Hen egg white lysozyme (HEWL; EC 3.2.1.17; 14,315 Da) was purchased from Sigma (Oakville, Ontario, Canada). Mutant E78C Bcx was prepared as described (35). The substrates 2,5-dinitrophenyl ␤-xylobioside (DNPX 2 ) and 2,4-dinitrophenyl ␤-cellobioside (DNPC) were prepared as described previously (20,30).…”

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confidence: 99%

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Hekmat¹,

Kim²,

Williams³

et al. 2005

Journal of Biological Chemistry

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“…Prior studies have used mutations that add or remove a sidechain methylene group to test the importance of positioning active-site carboxylate groups (19)(20)(21)(22)(23)(24). Such mutations are typically highly deleterious and have been shown to displace the position of the carboxylate by ∼1 Å relative to its wild-type position in several cases (19)(20)(21)(22)(23), a displacement similar to that observed for the Phe54Gly mutation described above.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 84%

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Schwans¹,

Sunden²,

Lassila³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogenbonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.enzyme catalysis | general-base catalysis | noncovalent interactions E nzymes use the same functional groups to achieve "chemical catalysis" as small-molecule catalysts, and yet enzymes attain much greater rate enhancements. A distinguishing feature of enzymes is that their reactions occur in highly specialized active sites that use noncovalent interactions to precisely position enzymatic functional groups relative to substrates and relative to other active-site features. Understanding how these groups are positioned within enzyme active sites is key for understanding the differences between enzymes and small-molecule catalysts.It is generally recognized that hydrophobic interactions can help bind and position substrates in enzyme active sites (1, 2). Beyond hydrophobic interactions, much attention has focused on the importance of hydrogen bonds in precisely positioning active-site groups directly involved in the chemical reaction (e.g., the catalytic triad in serine proteases), and the importance of hydrogen-bonding groups is supported by mutagenesis in many cases (e.g., refs. 1 and 3-5).In addition to hydrogen bonds, the cation-pi pair, formed between a side-chain or substrate cation and the negative electrostatic potential associated with the face of a pi system, has been widely discussed in hundreds of literature reports and has been shown to be important in both protein structure and proteinligand interactions (e.g., refs. 6 and 7). There has been much ...

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“…An alternative assignment strategy pH titrations of the histidine residues in BCX The chemical shifts of the IH, I3C, and ''N nuclei of histidines are dependent on the ionization state of the imidazole ring. Traditionally, this has provided a convenient avenue to measure experimentally the pK, values of histidine side chains in proteins (Markley, 1975;Blomberg et al, 1977 Lawson et al, 1996), this precluded the use of 'H-NMR to monitor the titrations of these two carbon bonded protons. An alternative method is to follow the titration of the histidines using the 'H-I3C HSQC experiment (Yu & Fesik, 1994).…”

Section: Assignment Of the Histidine Tautomeric Statesmentioning

confidence: 99%

Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange

et al. 1996

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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, I3C, and I5N 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 'HE' 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 low5 s-' at pH* 7.04 and 30 "C) is retarded by > 106-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at -6.5, as measured from the pH dependence of the "N-and 'H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N'*H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 "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.

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Effects of both Shortening and Lengthening the Active Site Nucleophile of Bacillus circulans Xylanase on Catalytic Activity

Cited by 57 publications

References 29 publications

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Active-site Peptide “Fingerprinting” of Glycosidases in Complex Mixtures by Mass Spectrometry

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Characterization of a buried neutral histidine residue inBacillus circulansxylanase: Nmr assignments, pH titration, and hydrogen exchange

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