Kinetic Mechanism and Inhibitor Characterization of WNK1 Kinase

Biochemistry ◽  
2009 ◽  
Vol 48 (43) ◽  
pp. 10255-10266 ◽  
Author(s):  
Yukiko I. Yagi ◽  
Koichi Abe ◽  
Kazunori Ikebukuro ◽  
Koji Sode
Keyword(s):  
Kinetic Mechanism

scholarly journals Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC

PLoS ONE ◽  
2011 ◽  
Vol 6 (9) ◽  
pp. e24951 ◽  
Author(s):  
Xiaolian Fan ◽  
Ilona Tkachyova ◽  
Ankit Sinha ◽  
Brigitte Rigat ◽  
Don Mahuran
Keyword(s):  
Mechanism Of Action ◽  
Kinetic Mechanism ◽  
Enzyme Deficient

Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism

FEBS Journal ◽  
2008 ◽  
Vol 275 (4) ◽  
pp. 775-787 ◽  
Author(s):  
Felix Stehle ◽  
Milton T. Stubbs ◽  
Dieter Strack ◽  
Carsten Milkowski
Keyword(s):  
Heterologous Expression ◽  
Kinetic Mechanism ◽  
Serine Carboxypeptidase

scholarly journals Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5

Biochemistry ◽  
2020 ◽  
Vol 59 (50) ◽  
pp. 4775-4786
Author(s):  
Alice R. Eddershaw ◽  
Christopher J. Stubbs ◽  
Lucy V. Edwardes ◽  
Elizabeth Underwood ◽  
Gregory R. Hamm ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Human Protein ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Protein Arginine Methyltransferase 5

Biochemical characterization of Mycobacterium tuberculosis IMP dehydrogenase: kinetic mechanism, metal activation and evidence of a cooperative system

RSC Advances ◽  
2014 ◽  
Vol 4 (50) ◽  
pp. 26271-26287 ◽  
Author(s):  
Diana Carolina Rostirolla ◽  
Thiago Milech de Assunção ◽  
Cristiano Valim Bizarro ◽  
Luiz Augusto Basso ◽  
Diogenes Santiago Santos
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biochemical Characterization ◽  
Kinetic Mechanism ◽  
Cooperative System ◽  
Imp Dehydrogenase ◽  
System P

Proposed kinetic mechanism for MtIMPDH in the presence of K+.

scholarly journals Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

2007 ◽  
Vol 189 (11) ◽  
pp. 3954-3959 ◽  
Author(s):  
Zhe Yang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Performance ◽  
Biochemical Characterization ◽  
Physiological Function ◽  
Catalytic Efficiency ◽  
Kinetic Mechanism ◽  
Arginine Catabolism ◽  
Ping Pong ◽  
Coupled Reaction

ABSTRACT The arginine transaminase (ATA) pathway represents one of the multiple pathways for l-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD+ and l-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V max and k cat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s−1. The apparent Km and catalytic efficiency (k cat/Km ) were 1.6 ± 0.1 mM and 24.1 mM−1 s−1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM−1 s−1 for l-arginine. When l-lysine was used as the substrate, MS analysis suggested Δ1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.

scholarly journals Kinetic mechanism and specificity of bovine milk sulphydryl oxidase

1984 ◽  
Vol 220 (1) ◽  
pp. 51-55 ◽  
Author(s):  
M X Sliwkowski ◽  
H E Swaisgood ◽  
D A Clare ◽  
H R Horton
Keyword(s):  
De Novo ◽  
Substrate Inhibition ◽  
Bovine Milk ◽  
Kinetic Mechanism ◽  
Enzyme Mechanism ◽  
Nitro Blue Tetrazolium ◽  
Blue Tetrazolium ◽  
Glutathione Oxidation ◽  
Metabolizing Enzyme

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

scholarly journals Purification and characterization of 5-ketofructose reductase from Erwinia citreus

1988 ◽  
Vol 253 (2) ◽  
pp. 511-516 ◽  
Author(s):  
J L Schrimsher ◽  
P T Wingfield ◽  
A Bernard ◽  
R Mattaliano ◽  
M A Payton
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Single Species ◽  
Kinetic Mechanism ◽  
Relative Molecular Mass ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Steady State Kinetic ◽  
State Kinetic

5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose.

scholarly journals Functional Characterization of a Novel ArgA from Mycobacterium tuberculosis

2005 ◽  
Vol 187 (9) ◽  
pp. 3039-3044 ◽  
Author(s):  
James C. Errey ◽  
John S. Blanchard
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Initial Velocity ◽  
Inhibitory Concentration ◽  
Functional Characterization ◽  
Initial Step ◽  
Kinetic Mechanism ◽  
Hill Coefficient ◽  
Arginine Biosynthesis ◽  
Inhibition Studies

ABSTRACT The Mycobacterium tuberculosis gene Rv2747 encodes a novel 19-kDa ArgA that catalyzes the initial step in l-arginine biosynthesis, namely the conversion of l-glutamate to α-N-acetyl-l-glutamate. Initial velocity studies reveal that Rv2747 proceeds through a sequential kinetic mechanism, with Km values of 280 mM for l-glutamine and 150 μM for acetyl-coenzyme A and with a k cat value of 200 min−1. Initial velocity studies with l-glutamate showed that even at concentrations of 600 mM, saturation was not observed. Therefore, only a k cat/Km value of 125 M−1 min−1 can be calculated. Inhibition studies reveal that the enzyme is strongly regulated by l-arginine, the end product of the pathway (50% inhibitory concentration, 26 μM). The enzyme was completely inhibited by 500 μM arginine, with a Hill coefficient of 0.60, indicating negatively cooperative binding of l-arginine.

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