scholarly journals Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

1971 ◽  
Vol 123 (5) ◽  
pp. 707-715 ◽  
Author(s):  
H. S. Bachelard ◽  
A. G. Clark ◽  
M. F. Thompson
Keyword(s):  
Reaction Mechanism ◽  
Enzymic Activity ◽  
Significant Inhibition ◽  
Kinetic Constants ◽  
Mitochondrial Activity ◽  
Kinetic Properties ◽  
Extraction And Purification ◽  
Dead End ◽  
Ping Pong ◽  
Atp Analogues

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP2−. Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP2− was the substrate varied. The Ki for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and βγ-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP2− was varied. AMP inhibition was competitive when MgATP2− was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.

The analysis of dead-end inhibition patterns for multisubstrate systems

1969 ◽  
Vol 47 (2) ◽  
pp. 91-108
Author(s):  
R. O. Hurst
Keyword(s):  
Reaction Mechanism ◽  
Rate Equation ◽  
Rate Constants ◽  
Additive Effect ◽  
Enzyme Form ◽  
Linear Fashion ◽  
Dead End ◽  
Ping Pong ◽  
Full Rate ◽  
Enzyme Species

The additive effect of a dead-end inhibitor combining in linear fashion with more than one of the enzyme species in a reaction mechanism is demonstrated. An equation for the calculation of the inhibitor constants that are required for the multicombinations of inhibitor from those obtained for the inhibitor combining with only one enzyme form is provided. A method of tabulation of the inhibitor constants with respect to the coefficients of the denominator terms of the full rate-equation for the uninhibited reaction is given, that facilitates the analysis of the inhibition patterns for the several inhibitor complexes that may be formed. The usefulness of the method for calculating the rate constants for a 'Ping Pong Bi Bi' mechanism is illustrated.

Alternative mechanism for a bimolecular nonsequential enzymic reaction

1969 ◽  
Vol 47 (2) ◽  
pp. 111-115 ◽  
Author(s):  
R. O. Hurst
Keyword(s):  
Reaction Mechanism ◽  
Product Inhibition ◽  
Alternative Mechanism ◽  
Enzymic Reaction ◽  
Dead End ◽  
Ping Pong ◽  
Inhibition Studies ◽  
Different Order ◽  
Inhibition Pattern

An enzymic reaction mechanism characterized as 'di-Uni Iso Ping Pong' which has the same product inhibition pattern as the 'Ping Pong Bi Bi' mechanism but a different order for the release of products is discussed. A basis for differentiating the two mechanisms by dead-end inhibition studies is given.

scholarly journals All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2807-2819 ◽  
Author(s):  
Friederike Klemke ◽  
Gabriele Beyer ◽  
Linda Sawade ◽  
Ali Saitov ◽  
Thomas Korte ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Enzymic Activity ◽  
Phosphoryl Group ◽  
Nitrogen Deprivation ◽  
Ping Pong ◽  
Nucleoside Triphosphates ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Promoter Studies

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with k cat = 48.2 s−1 at pH 7.5 and 28 °C and K M = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

scholarly journals Pyrolysis Kinetic Properties of Thermal Insulation Waste Extruded Polystyrene by Multiple Thermal Analysis Methods

Materials ◽  
2020 ◽  
Vol 13 (24) ◽  
pp. 5595
Author(s):  
Ang Li ◽  
Wenlong Zhang ◽  
Juan Zhang ◽  
Yanming Ding ◽  
Ru Zhou
Keyword(s):  
Reaction Mechanism ◽  
Thermal Insulation ◽  
Conversion Rate ◽  
Model Fitting ◽  
Pso Algorithm ◽  
Energy Utilization ◽  
Kinetic Properties ◽  
Insulation Material ◽  
Heating Rates ◽  
Model Free

Extruded polystyrene (XPS) is a thermal insulation material extensively applied in building systems. It has attracted much attention because of outstanding thermal insulation performance, obvious flammability shortcoming and potential energy utilization. To establish the reaction mechanism of XPS’s pyrolysis, thermogravimetric experiments were performed at different heating rates in nitrogen, and multiple methods were employed to analyze the major kinetics of pyrolysis. More accurate kinetic parameters of XPS were estimated by four common model-free methods. Then, three model-fitting methods (including the Coats-Redfern, the iterative procedure and masterplots method) were used to establish the kinetic model. Since the kinetic models established by the above three model-fitting methods were not completely consistent based on different approximations, considering the effect of different approximates on the model, the reaction mechanism was further established by comparing the conversion rate based on the model-fitting methods corresponding to the possible reaction mechanisms. Finally, the accuracy of the above model-fitting methods and Particle Swarm Optimization (PSO) algorithm were compared. Results showed that the reaction function g(α) = (1 − α)−1 − 1 might be the most suitable to characterize the pyrolysis of XPS. The conversion rate calculated by masterplots and PSO methods could provide the best agreement with the experimental data.

scholarly journals A kinetic study of rabbit muscle pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 223-236 ◽  
Author(s):  
S. Ainsworth ◽  
N. Macfarlane
Keyword(s):  
Reaction Mechanism ◽  
Kinetic Study ◽  
Pyruvate Kinase ◽  
Random Order ◽  
Experimental Results ◽  
Rabbit Muscle ◽  
Dead End ◽  
Inhibition Constants ◽  
Muscle Pyruvate Kinase ◽  
Kinetics Of

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg2+, form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

scholarly journals The kinetics of rat liver and heart mitochondrial β-hydroxybutyrate dehydrogenase

1979 ◽  
Vol 179 (3) ◽  
pp. 579-581 ◽  
Author(s):  
G A Tucker ◽  
A P Dawson
Keyword(s):  
Rat Liver ◽  
Rat Heart ◽  
Mitochondrial Membrane ◽  
Liver Mitochondria ◽  
Kinetic Constants ◽  
Kinetic Properties ◽  
Kinetic Mechanisms ◽  
Solubilized Enzyme ◽  
Beta Hydroxybutyrate ◽  
Kinetics Of

The kinetic mechanisms of the beta-hydroxybutyrate dehydrogenase from rat heart and liver mitochondria were investigated. Both enzymes, show an Ordered Bi Bi mechanism and there are no major differences in the kinetic constants. In both cases, the solubilized enzyme, re-activated with phosphatidylcholine, shows kinetic properties very similar to those of the enzyme bound to the mitochondrial membrane.

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism

2011 ◽  
Vol 175 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Hyo Je Cho ◽  
Ha Yeon Cho ◽  
Kyung Jin Kim ◽  
Myung Hee Kim ◽  
Si Wouk Kim ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Reaction Mechanism ◽  
Type Reaction ◽  
Ping Pong
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