scholarly journals Inhibition of Horseradish Peroxidase Activity by Boroxine Derivative, Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH]

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Jelena Ostojic ◽  
Safija Herenda ◽  
Semira Galijasevic ◽  
Borivoj Galic ◽  
Mladen Milos
Keyword(s):  
Activation Energy ◽  
Horseradish Peroxidase ◽  
Catalytic Mechanism ◽  
Competitive Inhibition ◽  
Targeted Cancer Therapy ◽  
Human Tumour ◽  
Kinetic Measurements ◽  
Human Tumour Cells ◽  
Steady State Kinetic ◽  
State Kinetic

Recently research shows that horseradish peroxidase, HRP, when combined with other compounds, is highly reactive toward different human tumour cells and that better understanding of catalytic mechanism and inhibition HPR could lead to a new targeted cancer therapy. Thus, the inhibition of HRP activity by dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was investigated for possible explanation of previously observed antitumour activities of this promising drug. HRP activity was studied under steady-state kinetic conditions by a spectrophotometric method. In the absence of the inhibitor values of Km = 0.47 mM and Vmax = 0.34 mM min−1, respectively, were determined. The hydrogen peroxide H2O2 kinetic measurements show a competitive inhibition with the inhibition constant KI = 2.56 mM. The activation energy Ea values were found to be very similar for both reactions; in the absence of inhibitor activation energy was 17.7 kJ mol−1 and in the presence of inhibitor activation energy was 16.3 kJ mol−1. The values of Arrhenius constants were found to be different; A = 4.635 s−1 was measured in the absence of inhibitor while in the presence of inhibitor Arrhenius constant was 1.745 s−1 showing that K2[B3O3F4OH] initiates conformational change in the structure of the HRP and subsequently reduces its activity.

scholarly journals Characterization of Nicotinamidases: Steady State Kinetic Parameters, Classwide Inhibition by Nicotinaldehydes, and Catalytic Mechanism

Biochemistry ◽  
2010 ◽  
Vol 49 (49) ◽  
pp. 10421-10439 ◽  
Author(s):  
Jarrod B. French ◽  
Yana Cen ◽  
Tracy L. Vrablik ◽  
Ping Xu ◽  
Eleanor Allen ◽  
...  
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Catalytic Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals Proton-linked l-rhamnose transport, and its comparison with l-fucose transport in Enterobacteriaceae

1993 ◽  
Vol 290 (3) ◽  
pp. 833-842 ◽  
Author(s):  
J A R Muiry ◽  
T C Gunn ◽  
T P McDonald ◽  
S A Bradley ◽  
C G Tate ◽  
...  
Keyword(s):  
Transport System ◽  
Erwinia Carotovora ◽  
Alkaline Ph ◽  
Transport Activity ◽  
Ph Change ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Competitive Inhibitors ◽  
State Kinetic

1. An alkaline pH change occurred when L-rhamnose, L-mannose or L-lyxose was added to L-rhamnose-grown energy-depleted suspensions of strains of Escherichia coli. This is diagnostic of sugar-H+ symport activity. 2. L-Rhamnose, L-mannose and L-lyxose were inducers of the sugar-H+ symport and of L-[14C]rhamnose transport activity. L-Rhamnose also induced the biochemically and genetically distinct L-fucose-H+ symport activity in strains competent for L-rhamnose metabolism. 3. Steady-state kinetic measurements showed that L-mannose and L-lyxose were competitive inhibitors (alternative substrates) for the L-rhamnose transport system, and that L-galactose and D-arabinose were competitive inhibitors (alternative substrates) for the L-fucose transport system. Additional measurements with other sugars of related structure defined the different substrate specificities of the two transport systems. 4. The relative rates of H+ symport and of sugar metabolism, and the relative values of their kinetic parameters, suggested that the physiological role of the transport activity was primarily for utilization of L-rhamnose, not for L-mannose or L-lyxose. 5. L-Rhamnose transport into subcellular vesicles of E. coli was dependent on respiration, was optimal at pH 7, and was inhibited by protonophores and ionophores. It was insensitive to N-ethylmaleimide or cytochalasin B. 6. L-Rhamnose, L-mannose and L-lyxose each elicited an alkaline pH change when added to energy-depleted suspensions of L-rhamnose-grown Salmonella typhimurium LT2, Klebsiella pneumoniae, Klebsiella aerogenes, Erwinia carotovora carotovora and Erwinia carotovora atroseptica. The relative rates of subsequent acidification varied, depending on both the organism and the sugar. L-Fucose promoted an alkaline pH change in all the L-rhamnose-induced organisms except the Erwinia species. No L-rhamnose-H+ symport occurred in any organism grown on L-fucose. 7. All these results showed that L-rhamnose transport into the micro-organisms occurred by a system different from that for L-fucose transport. Both systems are energized by the trans-membrane electrochemical gradient of protons. 8. Neither steady-state kinetic measurements nor binding-protein assays revealed the existence of a second L-rhamnose transport system in E. coli.

scholarly journals Dihydrolipoamide dehydrogenase from halophilic archaebacteria

1984 ◽  
Vol 218 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M J Danson ◽  
R Eisenthal ◽  
S Hall ◽  
S R Kessell ◽  
D L Williams
Keyword(s):  
Steady State ◽  
Iodoacetic Acid ◽  
Enzymic Activity ◽  
Evolutionary Significance ◽  
Dihydrolipoamide Dehydrogenase ◽  
Kinetic Measurements ◽  
Steady State Kinetic ◽  
Halophilic Archaebacteria ◽  
Eukaryotic Organisms ◽  
State Kinetic

Dihydrolipoamide dehydrogenase has been discovered in the halophilic archaebacteria for the first time. The enzyme from both classical and alkaliphilic halobacteria has been investigated. (1) The enzyme specifically catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+. Enzymic activity was optimal at 2 M-NaCl and was remarkably resistant to thermal denaturation. (2) The relative molecular masses (Mr) of the native enzyme from the various species of halobacteria were determined to be within the range 112000-120000. (3) The enzyme exhibited a hyperbolic dependence of catalytic activity on both dihydrolipoamide and NAD+ concentrations. From these steady-state kinetic measurements the dissociation constant (Ks) of dihydrolipoamide was determined to be 57 (+/- 5) microM. (4) The enzyme was only susceptible to inactivation by iodoacetic acid in the presence of its reducing ligands, dihydrolipoamide or NADH. The rate of inactivation followed a hyperbolic dependence on the concentration of dihydrolipoamide, from which the Ks of this substrate was calculated to be 55 (+/- 7) microM. Together with the steady-state kinetic data, the pattern of inactivations is consistent with the involvement in catalysis of a reversibly reducible disulphide bond, as has been found in dihydrolipoamide dehydrogenase from non-archaebacterial species. In eubacterial and eukaryotic organisms, dihydrolipoamide dehydrogenase functions in the 2-oxo acid dehydrogenase complexes. These multienzyme systems have not been detected in the archaebacteria, and, in the context of this apparent absence, the possible function and evolutionary significance of archaebacterial dihydrolipoamide dehydrogenase are discussed.

scholarly journals Orphan Macrodomain Protein (Human C6orf130) Is an O-Acyl-ADP-ribose Deacylase

2011 ◽  
Vol 286 (41) ◽  
pp. 35955-35965 ◽  
Author(s):  
Francis C. Peterson ◽  
Dawei Chen ◽  
Betsy L. Lytle ◽  
Marianna N. Rossi ◽  
Ivan Ahel ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Phylogenic Tree ◽  
Cellular Processes ◽  
Steady State Kinetic ◽  
Lysine Residues ◽  
Binding Cleft ◽  
And Function ◽  
State Kinetic

Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD+-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD+-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the β5-α4 loop that covers the bound ADPr, suggesting that the β5-α4 loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues.

Stopped-Flow, Pre-Steady-State Kinetic Study of Horseradish Peroxidase Catalysis in Nonaqueous Media

1996 ◽  
Vol 799 (1 Enzyme Engine) ◽  
pp. 364-375
Author(s):  
SHUNGUANG WANG ◽  
WEI LIU ◽  
XINSONG Ji ◽  
LIN MA ◽  
TIEJIN Li ◽  
...  
Keyword(s):  
Steady State ◽  
Horseradish Peroxidase ◽  
Kinetic Study ◽  
Stopped Flow ◽  
Nonaqueous Media ◽  
Steady State Kinetic ◽  
State Kinetic

Detection and characterization of VIM-52, a new variant of VIM-1 from Klebsiella pneumoniae clinical isolate

2021 ◽  
Author(s):  
Marie de Barsy ◽  
Paola Sandra Mercuri ◽  
Saoussen Oueslati ◽  
Eddy Elisée ◽  
Te-Din Huang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Substrate Specificity ◽  
Clinical Isolate ◽  
Targeted Mutagenesis ◽  
Kinetic Measurements ◽  
Steady State Kinetic ◽  
New Variant ◽  
State Kinetic ◽  
Global Health Problem

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

scholarly journals Inhibition of dimeric dihydrodiol dehydrogenases of rabbit and pig lens by ascorbic acid

1991 ◽  
Vol 275 (1) ◽  
pp. 121-126 ◽  
Author(s):  
A Hara ◽  
M Shinoda ◽  
T Kanazu ◽  
T Nakayama ◽  
Y Deyashiki ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Reverse Reaction ◽  
Ascorbate Oxidase ◽  
Binary Complex ◽  
Kinetic Measurements ◽  
Isoascorbic Acid ◽  
Dead End ◽  
Steady State Kinetic ◽  
Ic50 Values ◽  
State Kinetic

The dehydrogenase activity of dimeric dihydrodiol dehydrogenases (DD) purified from pig and rabbit lenses was inhibited by either L-ascorbic acid or its epimer, isoascorbic acid, at pH 7.5. Isoascorbate [IC50 (concn. giving 50% inhibition) = 0.043 mM for the pig enzyme; IC50 = 0.13 mM for the rabbit enzyme] was a more potent inhibitor than ascorbate (IC50 values 0.45 and 0.90 mM respectively), but 1 mM-dehydroascorbate gave less than 30% inhibition. Glucose, glucuronate, gulono-gamma-lactone, glutathione and dithiothreitol did not inhibit the enzyme activity. The inhibition by isoascorbate and ascorbate was instantaneous and reversible, and their inhibitory potency was decreased by addition of ascorbate oxidase. In the reverse reaction, isoascorbate and ascorbate gave low IC50 values of 0.013 and 0.10 mM respectively for the pig enzyme and 0.025 and 0.25 mM for the rabbit enzyme. The inhibition patterns by the two compounds were competitive with respect to dihydrodiols of naphthalene and benzene and uncompetitive with respect to NADP+, but those in the reverse reaction were uncompetitive with respect to both carbonyl substrate and NADPH. The steady-state kinetic measurements in the forward and reverse reactions by the pig enzyme were consistent with an ordered Bi Bi mechanism, in which NADP+ binds to the enzyme first and NADPH leaves last. The results indicate that ascorbate and its epimer directly bind to an enzyme: NADP+ binary complex as dead-end inhibitors. Thus ascorbate may be an important modulator of DD in the lens.

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