scholarly journals Properties and mechanism of action of pyruvate, phosphate dikinase from leaves

1969 ◽  
Vol 114 (1) ◽  
pp. 117-125 ◽  
Author(s):  
T. J. Andrews ◽  
M. D. Hatch
Keyword(s):  
Mechanism Of Action ◽  
Sugar Cane ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Reverse Direction ◽  
Pyruvate Phosphate Dikinase ◽  
Reaction Proceeds ◽  
Michaelis Constants ◽  
Phosphate Groups ◽  
Mechanism Of The Reaction

1. Sugar-cane leaf pyruvate,Pi dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and Pi into phosphoenolpyruvate, AMP and PPi. 2. The purified enzyme was stable at pH8·3 only if stored at about 20° in the presence of Mg2+ and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PPi were 0·11mm and 0·04mm respectively and that for AMP was less than 4μm. 4. At pH8·3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PPi were derived from Pi and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps: Enzyme+ATP+Pi ⇌ Enzyme–P+AMP+PPi Enzyme–P+pyruvate ⇌ Enzyme+phosphoenolpyruvate

scholarly journals Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

Properties of Phosphoenolpyruvate Carboxykinase Operative in C4 Pathway Photosynthesis

1977 ◽  
Vol 4 (2) ◽  
pp. 207 ◽  
Author(s):  
MD Hatch ◽  
S Mau
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Reverse Direction ◽  
Nucleoside Triphosphate ◽  
Dihydroxyacetone Phosphate ◽  
C4 Pathway ◽  
Phosphoglyceric Acid ◽  
Michaelis Constants ◽  
Chloris Gayana ◽  
Fructose 1,6 Bisphosphate ◽  
Chloris Gayana Kunth

A procedure is described for partially purifying phosphoenolpyruvate carboxykinase [ATP : oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from leaves of Chloris gayana Kunth. In three steps the enzyme was purified about 60-fold with 22% recovery of activity. This procedure removes enzymes, particularly malate dehydrogenase, that preclude the use of a simple spectrophotometric assay for phosphoenolpyruvate carboxykinase. The activity of the enzyme in the direction of oxaloacetate decarboxylation was about 10 times that in the reverse direction. At the optimal pH of 8.0, ATP was the preferred nucleoside triphosphate but CTP, UTP, GTP and ITP were also active. A requirement for Mn2+ could not be replaced by Mg2+. The Michaelis constants for oxaloacetate and ATP were 0.035 mM and 0.024 nM, respectively. The photosynthetic intermediates fructose 1,6-bisphosphate, 3-phosphoglyceric acid and dihydroxyacetone phosphate significantly inhibited the enzyme at concentrations in the region of 1-5 mM. Unlike the phosphoenolpyruvate carboxykinase from other sources, the capacity of the leaf enzyme to catalyse the decarboxylation of oxaloacetate to pyruvate was negligible. The properties of the enzyme are discussed in relation to its proposed role in C4 pathway photosynthesis.

scholarly journals A Mechanism of Action for Hydroxychloroquine and Azithromycin to Inhibit Coronavirus Disease COVID-19

2020 ◽  
Author(s):  
Charles Schaper
Keyword(s):  
Hydrogen Bond ◽  
Structural Biology ◽  
Mechanism Of Action ◽  
Adenine Nucleotide ◽  
Intermolecular Hydrogen Bond ◽  
Delivery Vector ◽  
Uracil Nucleotide ◽  
Phosphate Groups ◽  
End Group ◽  
Ionic Coupling

Hydroxychloroquine and azithromycin have clinical promise to treat COVID-19, although its mechanism of action to inhibit the replication of coronavirus is unclear. Using molecular modeling and recent discoveries made by this lab on the structure of nucleic acids, a mechanism of action is developed for hydroxychloroquine (HCQ) and azithromycin (AZR) to inhibit the replication of the coronavirus disease COVID-19. The mechanism involves: (1) binding the Cl end-element of HCQ through ionic means to adjacent phosphate groups of the uracil nucleotide; (2) forming an intermolecular hydrogen bond of an NH group of HCQ to an open oxygen element of uracil; (3) binding OH end group of HCQ through ionic means with adjacent phosphate groups of the adenine nucleotide. The mechanism of action is extended to AZR as a drug delivery vector that collects HCQ and two ions of positive two charge, such as Mg2+, Zn2+ or Ca2+, and delivers the assembly to a secondary structure of single-strand RNA. As with HCQ, the structural biology of AZR is compatible for use as a collection and delivery vesicle including: (1) open access for the Cl end element and the NH group of HCQ to align and bind with Uracil, and (2) the ability to deliver and bind through ionic coupling of the OH end group of HCQ to the adenine nucleotide. The molecular ionic attachment of HCQ to RNA nucleotides enabled by AZR results in the inhibition of the replication capability of the coronavirus disease COVID-19.

N-Heterocyclic Carbene-Catalyzed Synthesis of Ynones via C–H Alkynylation of Aldehydes with Alkynyliodonium Salts

2019 ◽  
Author(s):  
Adam A. Rajkiewicz ◽  
Natalia Wojciechowska ◽  
Marcin Kalek
Keyword(s):  
Density Functional ◽  
Bond Formation ◽  
Kinetic Studies ◽  
Density Functional Theory Calculations ◽  
Hypervalent Iodine ◽  
Functional Theory ◽  
Leaving Group ◽  
13C Labelling ◽  
Reaction Proceeds ◽  
Mechanism Of The Reaction

Alkynylation of aldehydes with alkynyl(aryl)iodonium salts catalyzed by an N-heterocyclic carbene (NHC) has been developed. The application of the organocatalyst and the hypervalent iodine group-transfer reagent allowed for metal-free C–H functionalization and C–C bond formation. The reaction proceeds under exceptionally mild conditions, at –40 ⁰C and in the presence of an amine base, providing access to an array of heteroaryl-propargyl ketones containing various substituents in good to excellent yields. The mechanism of the reaction was investigated by means of both experiments and density functional theory calculations. 13C-labelling and computations determined that the key alkynyl transfer step occurs via an unusual direct SN2 substitution of iodine-based leaving group by Breslow intermediate nucleophile at an acetylenic carbon. Moreover, kinetic studies revealed that the turnover-limiting step of the catalytic cycle is the generation of the Breslow intermediate, whereas the subsequent C–C bond-formation is a fast process. These results were fully reproduced and rationalized by the computed full free energy profile of the reaction, showing that the largest energy span is located between protonated NHC and the transition state for the carbene attack on the aldehyde substrate.<br>

Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by oxygen-18 isotope exchange

Biochemistry ◽  
1991 ◽  
Vol 30 (34) ◽  
pp. 8441-8453 ◽  
Author(s):  
Lawrence J. Hyland ◽  
Thaddeus A. Tomaszek ◽  
Gerald D. Roberts ◽  
Steven A. Carr ◽  
Victoria W. Magaard ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Kinetic Characterization ◽  
Reaction Intermediates ◽  
Immunodeficiency Virus ◽  
Human Immunodeficiency Virus 1

scholarly journals Kinetic properties of spermine synthase from bovine brain

1983 ◽  
Vol 215 (3) ◽  
pp. 669-676 ◽  
Author(s):  
R L Pajula
Keyword(s):  
Initial Velocity ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Bovine Brain ◽  
Kinetic Properties ◽  
Methylthioadenosine Phosphorylase ◽  
Spermine Synthase ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Competitive Product

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5′-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5′-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5′-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

scholarly journals Purification and catalytic properties of l-valine dehydrogenase from Streptomyces cinnamonensis

1989 ◽  
Vol 261 (3) ◽  
pp. 853-861 ◽  
Author(s):  
N D Priestley ◽  
J A Robinson
Keyword(s):  
Initial Velocity ◽  
Reductive Amination ◽  
Catalytic Properties ◽  
Product Inhibition ◽  
Aminobutyric Acid ◽  
Oxidative Deamination ◽  
Streptomyces Cinnamonensis ◽  
Michaelis Constants ◽  
Pyrimidine Bases ◽  
Inhibition Studies

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4′ of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.

scholarly journals The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

1973 ◽  
Vol 135 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Brian Gillham
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Glutathione S Transferase ◽  
Michaelis Constant ◽  
Dead End ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).

scholarly journals Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase

1976 ◽  
Vol 157 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J W London ◽  
L M Shaw ◽  
D Fetterolf ◽  
D Garfinkel
Keyword(s):  
Initial Velocity ◽  
Competitive Inhibition ◽  
Linear Regression Analysis ◽  
Kinetic Constants ◽  
Reaction Products ◽  
Gamma Glutamyl ◽  
Ping Pong ◽  
Donor Acceptor ◽  
Michaelis Constants ◽  
Hog Kidney

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.

Eco-friendly Suzuki–Miyaura coupling of arylboronic acids to aromatic ketones catalyzed by the oxime-palladacycle in biosolvent 2-MeTHF

2016 ◽  
Vol 40 (4) ◽  
pp. 3119-3123 ◽  
Author(s):  
Manoj Mondal ◽  
Utpal Bora
Keyword(s):  
Green Chemistry ◽  
Sugar Cane ◽  
Coupling Reaction ◽  
Cross Coupling ◽  
Aromatic Ketones ◽  
Acyl Chlorides ◽  
Arylboronic Acids ◽  
Cross Coupling Reaction ◽  
Reaction Proceeds ◽  
Aryl Ketones

Oxime-palladacycle catalyzed aerobic cross-coupling reaction of arylboronic acids and acyl chlorides to yield aryl ketones was developed. The reaction proceeds efficiently in 2-MeTHF, which is derived from corncobs, sugar cane, and fulfils the 3rd, 5th and 7th principles of Green Chemistry.

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