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 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 Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

1999 ◽  
Vol 341 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Donna D. SONG ◽  
Nicholas A. JACQUES
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Streptococcus Salivarius ◽  
Acetobacter Diazotrophicus ◽  
E Coli ◽  
Ping Pong ◽  
Mechanism Of Catalysis ◽  
Enzyme Intermediate

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 °C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

.beta.-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin

Biochemistry ◽  
1980 ◽  
Vol 19 (13) ◽  
pp. 2895-2901 ◽  
Author(s):  
Jed Fisher ◽  
Joel G. Belasco ◽  
Sundeep Khosla ◽  
Jeremy R. Knowles
Keyword(s):  
Escherichia Coli ◽  
Beta Lactamase ◽  
Enzyme Intermediate

scholarly journals Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli

1988 ◽  
Vol 250 (3) ◽  
pp. 789-796 ◽  
Author(s):  
P N Lowe ◽  
S Rhodes
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
E Coli ◽  
Step Procedure ◽  
Ping Pong ◽  
Protein Acetyltransferase ◽  
Enzyme Intermediate ◽  
Specific Reagent

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

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