Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus faecalis

1988 ◽  
Vol 66 (1) ◽  
pp. 76-80 ◽  
Author(s):  
E. B. Waygood ◽  
K. Pasloske ◽  
L. T. J. Delbaere ◽  
J. Deutscher ◽  
W. Hengstenberg
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Phosphotransferase System ◽  
Temperature Dependent ◽  
Streptococcus Faecalis ◽  
Terminal Residue ◽  
Hydrolysis Of

The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1- phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2020 ◽  
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M. N. Scapin ◽  
William de Andrade ◽  
Maira Fasciotti ◽  
Mariana T. Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Lymphoblastic Leukemia ◽  
Biophysical Characterization ◽  
E Coli ◽  
Terminal Loop ◽  
Hydrolysis Of

AbstractThe hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli

Biochemistry ◽  
1991 ◽  
Vol 30 (40) ◽  
pp. 9601-9607 ◽  
Author(s):  
J. William Anderson ◽  
Pradeep Bhanot ◽  
Fawzey Georges ◽  
Rachel E. Klevit ◽  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Phosphotransferase System ◽  
Terminal Residue ◽  
Carboxy Terminal

scholarly journals Charge Reversal of a Critical Active-Site Residue of Cytochrome-c Peroxidase. Characterization of the Arg48Glu Variant

1997 ◽  
Vol 243 (1-2) ◽  
pp. 72-84 ◽  
Author(s):  
Jordi Bujons ◽  
Alexander Dikiy ◽  
Juan C. Ferrer ◽  
Lucia Banci ◽  
A. Grant Mauk
Keyword(s):  
Cytochrome C ◽  
Active Site ◽  
Cytochrome C Peroxidase ◽  
Active Site Residue ◽  
Charge Reversal

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

2006 ◽  
Vol 34 (1) ◽  
pp. 143-145 ◽  
Author(s):  
T.A. Clarke ◽  
A.M. Hemmings ◽  
B. Burlat ◽  
J.N. Butt ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Desulfovibrio Desulfuricans ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Wolinella Succinogenes ◽  
Nitrite Reductases ◽  
Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

Characterization of Escherichia coli thioredoxins with altered active site residues

Biochemistry ◽  
1990 ◽  
Vol 29 (15) ◽  
pp. 3701-3709 ◽  
Author(s):  
Florence K. Gleason ◽  
Chang Jin Lim ◽  
Maryam Gerami-Nejad ◽  
James A. Fuchs
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Site Residues

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

scholarly journals Molecular Characterization of Cycloinulooligosaccharide Fructanotransferase from Bacillus macerans

2001 ◽  
Vol 67 (2) ◽  
pp. 995-1000 ◽  
Author(s):  
Hwa-Young Kim ◽  
Yong-Jin Choi
Keyword(s):  
Escherichia Coli ◽  
Thermal Stability ◽  
Striking Difference ◽  
Bacillus Macerans ◽  
Specific Manner ◽  
Specificity Constant ◽  
Processing Event ◽  
Hydrolysis Of ◽  
Thermal Stability Of

ABSTRACT Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of β-(2→1)-linkedd-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculatedM r of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (k cat/Km ) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

scholarly journals An active-site peptide from pepsin C

1971 ◽  
Vol 123 (1) ◽  
pp. 75-82 ◽  
Author(s):  
J. Kay ◽  
A. P. Ryle
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Ph Values ◽  
Complete Inactivation ◽  
Active Site Sequence

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.

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