scholarly journals Modification of catalytically important carboxy residues in endoglucanase D from Clostridium thermocellum

1992 ◽  
Vol 285 (1) ◽  
pp. 319-324 ◽  
Author(s):  
P Tomme ◽  
J van Beeumen ◽  
M Claeyssens
Keyword(s):  
Active Site ◽  
Clostridium Thermocellum ◽  
Ph Dependence ◽  
Reversed Phase ◽  
Spectrophotometric Analysis ◽  
Active Site Residues ◽  
Terminal Sequence ◽  
Local Conservation ◽  
Woodward’S Reagent K ◽  
Phase Liquid

Endoglucanase D (EC 3.2.1.4; EGD) from Clostridium thermocellum is rapidly (k = 216 M-1.min-1) and almost completely (greater than 95%) inactivated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulphonate). Spectrophotometric analysis at 340 nm reveals that eight carboxy residues react, whereas specific ligands protect one residue against modification. The enzyme retains it full activity under the latter conditions. The kinetics and pH-dependence of inactivation point towards the involvement of one or more essential carboxy groups with a pKa of 5.7-5.8. Samples modified in the absence or presence of ligand were analysed by reversed-phase liquid chromatography after proteolysis with subtilisin. Dual-wavelength monitoring at 214 and 340 nm during this fractionation leads to the identification of a putatively active-site peptide (Gly-508-Ala-562) which was further characterized by amino acid and partial N-terminal sequence analyses. Asp-546 and Glu-555 are postulated as possible active-site residues. This follows from alignments using ten endoglucanase sequences belonging to the same family. Strong local conservation suggests that this C-terminal sequence is structurally and/or functionally important.

scholarly journals Functional Comparison of the Two Bacillus anthracis Glutamate Racemases

2007 ◽  
Vol 189 (14) ◽  
pp. 5265-5275 ◽  
Author(s):  
Dylan Dodd ◽  
Joseph G. Reese ◽  
Craig R. Louer ◽  
Jimmy D. Ballard ◽  
M. Ashley Spies ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Active Site ◽  
Genomic Sequence ◽  
Ph Dependence ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Active Site Residues ◽  
Glutamate Racemase ◽  
Genes Encoding ◽  
Exclusion Chromatography

ABSTRACT Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of l-glutamate and d-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of l-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features.

Reversed-phase liquid-chromatographic analysis of hemodialysate from uremic patients.

1976 ◽  
Vol 22 (9) ◽  
pp. 1522-1527 ◽  
Author(s):  
F C Senftleber ◽  
A G Halline ◽  
H Veening ◽  
D A Dayton
Keyword(s):  
High Performance ◽  
Reversed Phase ◽  
Spectrophotometric Analysis ◽  
Artificial Kidney ◽  
Efficient Separation ◽  
Liquid Chromatographic Analysis ◽  
Phase Liquid ◽  
Uremic Patients ◽  
Chromatographic Peaks ◽  
Liquid Chromatographic

Abstract With reversed-phase high-performance liquid chromatography we effected rapid and efficient separation of metabolites present in hemodialysate fluid from uremic patients on the artificial kidney. With an aqueous sodium acetate/methanol gradient as mobile phase, more than 50 ultraviolet-absorbing constituents were resolved in a 70-min chromatogram of a 100-mul sample of dialysate. Many of the metabolites could be detected in concentrations below 0.1 mg/liter. From results of ultraviolet spectrophotometric analysis and the gas- and liquid-chromatographic properties of standards, half the observed chromatographic peaks have been characterized or identified. The method has been shown to be more than 10-fold faster than comparable separations by ion-exchange techniques.

4-Oxalocrotonate Tautomerase:  pH Dependence of Catalysis and pKaValues of Active Site Residues†

Biochemistry ◽  
1996 ◽  
Vol 35 (3) ◽  
pp. 814-823 ◽  
Author(s):  
James T. Stivers ◽  
Chitrananda Abeygunawardana ◽  
Albert S. Mildvan ◽  
Gholamhossein Hajipour ◽  
Christian P. Whitman
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Active Site Residues

Effects of Mutations of the Active Site Arginine Residues in 4-Oxalocrotonate Tautomerase on the pKaValues of Active Site Residues and on the pH Dependence of Catalysis†

Biochemistry ◽  
1999 ◽  
Vol 38 (38) ◽  
pp. 12358-12366 ◽  
Author(s):  
Robert M. Czerwinski ◽  
Thomas K. Harris ◽  
William H. Johnson, ◽  
Patricia M. Legler ◽  
James T. Stivers ◽  
...  
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Active Site Residues ◽  
Arginine Residues

scholarly journals Characterization of C-S Lyase fromC. diphtheriae: A Possible Target for New Antimicrobial Drugs

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Alessandra Astegno ◽  
Alejandro Giorgetti ◽  
Alessandra Allegrini ◽  
Barbara Cellini ◽  
Paola Dominici
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
Ph Dependence ◽  
Amino Acid Replacement ◽  
Site Directed Mutagenesis ◽  
Microbial Pathogens ◽  
Active Site Residues ◽  
Methionine Biosynthesis ◽  
Antimicrobial Drugs

The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase fromCorynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein inE. coliand analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin fromTreponema denticolaindicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site ofC. diphtheriaeC-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics.

scholarly journals Asymmetric protonation of EmrE

2015 ◽  
Vol 146 (6) ◽  
pp. 445-461 ◽  
Author(s):  
Emma A. Morrison ◽  
Anne E. Robinson ◽  
Yongjia Liu ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Active Site ◽  
Nmr Spectra ◽  
Ph Dependence ◽  
Conformational Exchange ◽  
Coupling Mechanism ◽  
Active Site Residues ◽  
Spectra Analysis ◽  
Pka Values ◽  
Small Multidrug Resistance

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with 1H-15N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.

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