scholarly journals Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids

1988 ◽  
Vol 250 (3) ◽  
pp. 917-920 ◽  
Author(s):  
J M Breitenbach ◽  
R P Hausinger
Keyword(s):  
Boric Acid ◽  
Proteus Mirabilis ◽  
Ph Dependence ◽  
Competitive Inhibitor ◽  
Boronic Acids ◽  
Urinary Stones ◽  
Partial Purification ◽  
Pka Values

Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively.

scholarly journals Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity

2021 ◽  
Vol 22 (13) ◽  
pp. 7205
Author(s):  
Matheus V. C. Grahl ◽  
Augusto F. Uberti ◽  
Valquiria Broll ◽  
Paula Bacaicoa-Caruso ◽  
Evelin F. Meirelles ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Stone Formation ◽  
Cell Types ◽  
Bioinformatic Analysis ◽  
Urinary Stone ◽  
Enzymatic Properties ◽  
Hek293 Cells ◽  
Urinary Stones ◽  
Isolated Cells ◽  
Tnf Α

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures’ supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1β and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1β. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

scholarly journals Anomalous pH-dependence of the activity of human matrilysin (matrix metalloproteinase-7) as revealed by nitration and amination of its tyrosine residues

2005 ◽  
Vol 386 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Yuko MUTA ◽  
Hiroshi ONEDA ◽  
Kuniyo INOUYE
Keyword(s):  
Matrix Metalloproteinase ◽  
Strong Influence ◽  
Negative Charge ◽  
Ph Dependence ◽  
Michaelis Constant ◽  
Pka Values ◽  
Tyrosine Residues ◽  
Alkaline Side ◽  
Matrix Metalloproteinase 7

Matrilysin activity exhibits a broad bell-shaped pH-dependence profile, with pKa values of 4.0 and 9.8. A maximum of five out of eight tyrosine residues in matrilysin were nitrated with tetranitromethane. On nitration of between one and five tyrosines, pKa at the alkaline side (pKe2) was shifted from 9.8 to 10.3–10.6, while that at the acidic side (pKe1) was not altered. The pKe2 that was shifted by nitration to 10.3–10.6 was restored to 9.4–9.7 by subsequent amination, suggesting that the shift in pKe2 is induced by a negative charge introduced on the most reactive tyrosine, Tyr-150. The Michaelis constant (Km) observed at pH 10 was decreased by nitration as a result of the increase in pKe2, suggesting that the residue with pKe2 may play a role in the recognition of substrate. When four or five tyrosines were nitrated, the activity at pH <7 decreased significantly, while that at pH 7–10 was unchanged, and thus the pH-dependence was not bell-shaped, but anomalous, with a third pKa (pKe3) of 6.2–6.4 in addition to pKe1 and pKe2. This suggests the possibility that a newly introduced nitrotyrosine residue has a strong influence on the activity as an ionizable group.

scholarly journals Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters

1998 ◽  
Vol 330 (1) ◽  
pp. 311-314 ◽  
Author(s):  
F. RAMÓN ◽  
M. P. CASTILLÓN ◽  
I. DE LA MATA ◽  
C. ACEBAL
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Ph Dependence ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Acid Oxidase ◽  
Protonation State ◽  
Amino Acid Oxidase ◽  
Basic Group ◽  
Rhodotorula Gracilis

The variation of kinetic parameters of D-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. D-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the β-carboxylic group of the inhibitor.

scholarly journals Chemical synthesis and papain-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide

1985 ◽  
Vol 226 (2) ◽  
pp. 601-606 ◽  
Author(s):  
N E Mackenzie ◽  
J P G Malthouse ◽  
A I Scott
Keyword(s):  
Chemical Synthesis ◽  
Ph Dependence ◽  
Low Ph ◽  
Pka Values ◽  
Tetrahedral Intermediate ◽  
Ph Values ◽  
Km Value ◽  
Hydrolysis Of

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.

scholarly journals Boric acid and boronic acids inhibition of pigeonpea urease

2006 ◽  
Vol 21 (4) ◽  
pp. 467-470 ◽  
Author(s):  
K. Ravi Charan Reddy ◽  
Arvind M. Kayastha
Keyword(s):  
Boric Acid ◽  
Boronic Acids

Kinetic Studies of Platelet Aggregation Induced by Adenosine Diphosphate and Its Inhibition by Chelating Agents, Guanidino Compounds, and Adenosine

1967 ◽  
Vol 18 (03/04) ◽  
pp. 713-725 ◽  
Author(s):  
L Skoza ◽  
Marjorie B Zucker ◽  
Z Jerushalmy ◽  
R Grant
Keyword(s):  
Platelet Aggregation ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Adenosine Diphosphate ◽  
Competitive Inhibitor ◽  
Ionized Calcium ◽  
Guanidino Compounds ◽  
Lag Period ◽  
A Chain ◽  
Citrate Dextrose

SummaryThe kinetics, activation, pH dependence and inhibition of platelet aggregation induced by ADP were studied by recording the OD of stirred PRP from blood anticoagulated with acid-citrate-dextrose solution. There was a lag period between the addition of ADP and the initiation of aggregation. Addition of ADP in the absence of stirring or ionized calcium did not cause aggregation. When aggregation was subsequently initiated by stirring or the addition of CaCl2 there was no lag period. The steepest slope of the OD curve was used as the reaction velocity (V). It was affected by the concentration of calcium ions and was maximum at pH 8.0. When the ADP concentration was varied in the presence of different concentrations of ionized calcium, the overall kinetics revealed a series of rectangular hyperbola segments which did not pass through the origin. These observations led to the conclusion that the overall reaction can be depicted as a chain reaction: ADP interacts reversibly with platelets; when calcium is present these platelets become “sticky” and, when stirred, they aggregate. Kinetic studies of inhibition indicated that adenosine is a competitive inhibitor of ADP. The guanidino compounds tested were noncompetitive with respect to ADP but their inhibitory activity was counteracted by calcium.

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