scholarly journals Identification of Glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS

1998 ◽  
Vol 335 (2) ◽  
pp. 449-455 ◽  
Author(s):  
David J. VOCADLO ◽  
Lloyd F. MACKENZIE ◽  
Shouming HE ◽  
Gregory J. ZEIKUS ◽  
Stephen G. WITHERS
Keyword(s):  
Neutral Loss ◽  
Enzyme Inactivation ◽  
Peptide Sequence ◽  
Glycosyl Hydrolases ◽  
Inactivation Rate Constant ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dependent Inactivation ◽  
Peptic Digestion ◽  
Covalent Intermediate ◽  
Enzyme Intermediate

Thermoanaerobacterium saccharolyticum β-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining β-d-xylosidases and α-l-iduronidases. T. saccharolyticum β-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl–enzyme intermediate. Incubation of T. saccharolyticum β-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.089 min-1, dissociation constant for the inactivator Ki = 65 µM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-xylosyl–enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-α-d-xylosyl–enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-α-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.

scholarly journals Identification of Glu-519 as the catalytic nucleophile in β-mannosidase 2A from Cellulomonas fimi

2000 ◽  
Vol 351 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Dominik STOLL ◽  
Shouming HE ◽  
Stephen G. WITHERS ◽  
R. Antony J. WARREN
Keyword(s):  
Peptide Bond ◽  
Enzyme Inactivation ◽  
Ionization Mass ◽  
Glycosyl Hydrolases ◽  
Cellulomonas Fimi ◽  
Inactivation Rate Constant ◽  
Dependent Inactivation ◽  
The Difference ◽  
The Masses ◽  
Covalent Intermediate

Incubation of the β-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-β-d-mannosyl fluoride (2FManβF) resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.57min-1, dissociation constant for the inactivator Ki = 0.41mM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-mannosyl–β-mannosidase 2A (2FMan–Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glcβ(1–6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan–Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan–Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517–528 and 520–528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan–Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519–Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a β-mannosidase.

Synthesis of 5-fluoro-β-D-glucopyranosyluronic acid fluoride and its evaluation as a mechanistic probe of Escherichia coli β-glucuronidase

2001 ◽  
Vol 79 (5-6) ◽  
pp. 510-518 ◽  
Author(s):  
Alexander W Wong ◽  
Shouming He ◽  
Stephen G Withers
Keyword(s):  
Escherichia Coli ◽  
Triple Quadrupole Mass Spectrometer ◽  
Acid Fluoride ◽  
Radical Bromination ◽  
Dependent Inactivation ◽  
Phenacyl Ester ◽  
Peptic Digestion ◽  
Good Potential ◽  
Mechanistic Probe ◽  
Enzyme Intermediate

Synthesis of the potential mechanism-based inactivator of β-D-glucuronidases (5-fluoro-β-D-glucopyranosyluronic acid fluoride) was accomplished via a six-step process from D-glucuronic acid that involved radical bromination at C-5 and displacement of the bromide by fluoride. A key step in this process was the masking of the carboxylic acid as a phenacyl ester. This group is uniquely stable to conditions of photobromination and fluoride displacement, yet removable under very mild conditions. Incubation of the Escherichia coli β-glucuronidase with 5-fluoro-β-D-glucopyranosyluronic acid fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 5-fluoro-α-D-glucopyranosyluronic acid-enzyme. Peptic digestion of the 5-fluoro-α-D-glucopyranosyluronic acid-enzyme intermediate and subsequent analysis by liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer indicated the presence of a 5-fluoro-α-D-glucopyranosyluronic acid-modified peptide. This peptide was partially purified by HPLC and its sequence determined by tandem mass spectrometry in the daughter ion scan mode, permitting the identification of Glu504 as the catalytic nucleophile within the sequence ITEYGVD. This new reagent is therefore useful for the specific, mechanism-based inactivation of glycuronidases and has good potential in other studies of enzymes of this general class.Key words: β-glucuronidase, catalytic nucleophile, 5-fluoro-β-D-glucopyranosyluronic acid fluoride, electrospray MS.

scholarly journals The substrate/product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485

2012 ◽  
Vol 448 (3) ◽  
pp. 401-407 ◽  
Author(s):  
Chun-Hsiang Huang ◽  
Yu Sun ◽  
Tzu-Ping Ko ◽  
Chun-Chi Chen ◽  
Yingying Zheng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Product Inhibition ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Binding Modes ◽  
Glycosyl Hydrolases ◽  
Core Domain ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Cazy Database ◽  
Binding Cleft

Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48–2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel β-helix (residues 1–75 and 201–638) and the flanking region (residues 76–200) folds into a β-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.

scholarly journals KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

2020 ◽  
pp. jbc.RA120.015050
Author(s):  
Shrenik C Mehta ◽  
Ian M Furey ◽  
Orville A Pemberton ◽  
David M Boragine ◽  
Yu Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Common ◽  
Covalent Intermediate ◽  
Hydrolysis Of ◽  
Carbapenem Antibiotic ◽  
Enzyme Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

Measurement of alkaline phosphatase activity: characterization and identification of an inactivator in 2-amino-2-methyl-1-propanol.

1981 ◽  
Vol 27 (8) ◽  
pp. 1401-1409 ◽  
Author(s):  
R Rej ◽  
J P Bretaudiere ◽  
R W Jenny ◽  
K Y Jackson
Keyword(s):  
Alkaline Phosphatase ◽  
High Performance ◽  
Divalent Metal ◽  
Enzyme Inactivation ◽  
Time Dependent ◽  
Divalent Metal Ions ◽  
Molar Basis ◽  
Dependent Inactivation ◽  
Layer Chromatography ◽  
Absorbance Spectra

Abstract An inactivator of alkaline phosphatase (EC 3.1.3.1) in 2-amino-2-methyl-1-propanol is demonstrated and characterized. This time-dependent inactivation results from chelation of enzyme-bound Zn2+; it is reversed by addition of Zn2+ and, to a lesser extent, other divalent metal ions. Cu2+ is an effective spectral indicator and can be used to determine the presence and quantity of inactivator. Data obtained from enzyme inactivation, Cu2+ absorbance spectra, "high-performance" liquid chromatography, thin-layer chromatography, Fourier-transform infrared spectroscopy, and mass spectroscopy indicate that the inactivator is 5-amino-3-aza-2,2,5-trimethylhexanol. This compound, even in trace amounts (less than 0.05% on a molar basis), shown to inactivate alkaline phosphatase.

scholarly journals The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast

1993 ◽  
Vol 293 (1) ◽  
pp. 75-81 ◽  
Author(s):  
H Angliker ◽  
P Wikstrom ◽  
E Shaw ◽  
C Brenner ◽  
R S Fuller
Keyword(s):  
Rate Constant ◽  
Potent Inhibitor ◽  
Protein Processing ◽  
The Other ◽  
Peptide Sequence ◽  
Cleavage Sites ◽  
Precursor Processing ◽  
Inactivation Rate Constant ◽  
Potential Inhibitors ◽  
Kinetics Of

Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of ‘control’ proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase.

scholarly journals Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues

2004 ◽  
Vol 378 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Krisztina PAAL ◽  
Makoto ITO ◽  
Stephen G. WITHERS
Keyword(s):  
Kinetic Studies ◽  
Acid Base ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Dependent Inactivation ◽  
Paenibacillus Sp ◽  
Continuous Assay ◽  
Reduced Activity ◽  
Enzyme Intermediate

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the β-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-Nitrophenyl β-d-glucopyranoside [Km=0.27±0.02 mM and kcat/Km=(2.1±0.2)×106 M−1·s−1] and 2,4-dinitrophenyl β-d-glucopyranoside [Km=0.16±0.02 mM and kcat/Km=(2.9±0.4)×106 M−1·s−1] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/Km) on pH revealed a dependence on a group of pKa≤7.8 in the enzyme–substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside caused time-dependent inactivation (Ki=2.4±0.7 mM and ki=0.59±0.05 min−1) due to the accumulation of a trapped glycosyl–enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (>104), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product.

scholarly journals Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB

1998 ◽  
Vol 336 (1) ◽  
pp. 139-145 ◽  
Author(s):  
David L. ZECHEL ◽  
Shouming HE ◽  
Claude DUPONT ◽  
Stephen G. WITHERS
Keyword(s):  
Catalytic Domain ◽  
Neutral Loss ◽  
Competitive Inhibitor ◽  
Streptomyces Lividans ◽  
Acid Base ◽  
Anomeric Configuration ◽  
High Concentrations ◽  
Singly Charged ◽  
Kinetics Of ◽  
Enzyme Intermediate

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 Å resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid–base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032–16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2´,4´-dinitrophenyl 2-deoxy-2-fluoro-β-d-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact = 0.29±0.02 min-1 and Kinact = 0.72±0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki = 9±1 mM), a behaviour that was also observed with the substrate 2´,4´-dinitrophenyl β-d-cellobioside (kcat = 40±1 s-1, Km = 0.35±0.03 mM, Ki = 24±4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O = 0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact = 0.024 min-1, Kreact = 54 mM). Digestion of the labelled enzyme by pepsin followed by LC–ESI–tandem MS (MS–MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI–MS–MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid–base catalyst Glu-203 are conserved in all known family-12 sequences.

Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile

2001 ◽  
Vol 355 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Josep-Lluís VILADOT ◽  
Francesc CANALS ◽  
Xavier BATLLORI ◽  
Antoni PLANAS
Keyword(s):  
Laser Desorption ◽  
Hydrolytic Activity ◽  
Dependent Manner ◽  
Laser Desorption Ionization ◽  
Concentration Dependent Manner ◽  
Ionization Time ◽  
Glycosyl Donor ◽  
Covalent Intermediate ◽  
Enzyme Intermediate ◽  
Matrix Assisted Laser

The mutant E134A 1,3-1,4-β-glucanase from Bacilluslicheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner. A long-lived α-glycosyl formate is detected and identified by 1H-NMR and matrix-assisted laser desorption ionization–time-of-flight-MS. The intermediate is kinetically competent, since it is, at least partially, enzymically hydrolysed, and able to act as a glycosyl donor in transglycosylation reactions. This transient compound represents a true covalent glycosyl-enzyme intermediate mimic of the proposed covalent intermediate in the reaction mechanism of retaining glycosidases.

Reversible ATP-dependent inactivation of glycerophosphate acyltransferase from rat adipose tissue

1989 ◽  
Vol 67 (1) ◽  
pp. 48-52 ◽  
Author(s):  
M. Walsh ◽  
V. Durocher ◽  
A. Rodriguez
Keyword(s):  
Adipose Tissue ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Lipid Synthesis ◽  
Enzyme Inactivation ◽  
Glycerol Phosphate ◽  
Dependent Inactivation ◽  
Dependent Protein ◽  
Rat Adipose Tissue ◽  
Synthesis Regulation

Microsomal glycerophosphate acyltransferase from rat adipose tissue is shown to be inactivated with time upon incubation with ATP. The inactivation can be observed in postmitochondrial supernatant as well as in washed microsomes. However, the effect is more pronounced upon addition of the cytosolic fraction. This activity is specific for ATP, is dependent on the nucleotide concentration, and is prevented when ATP is substituted by β,γ-methylene-ATP. Some protection is provided by amiloride but not by EGTA or cAMP-protein kinase inhibitor. Also, the level of enzyme inactivation is not modified by addition of cAMP-dependent protein kinase and its substrates. Inactivated glycerol-phosphate acyltransferase from ATP-treated microsomes can be reactivated by incubation with partially purified protein phosphatase from rat liver. These results suggest the existence in adipose tissue of a protein kinase (cAMP independent) that may be involved in the regulation of glycerophosphate acyltransferase.Key words: glycerophosphate acyltransferase, lipid synthesis regulation, adipose tissue, protein kinase.

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