scholarly journals Kinetic studies on the two common inherited forms of human erythrocyte adenylate kinase

1972 ◽  
Vol 130 (3) ◽  
pp. 805-811 ◽  
Author(s):  
C. Brownson ◽  
N. Spencer
Keyword(s):  
Human Erythrocyte ◽  
Adenylate Kinase ◽  
Competitive Inhibition ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Product Inhibition ◽  
Reverse Direction ◽  
Kinetic Properties ◽  
Ping Pong ◽  
Variable Substrate

1. The kinetic properties of two genetic variants of human erythrocyte adenylate kinase were studied at limiting concentrations of both ADP and MgADP- in the forward direction and at limiting concentrations of both AMP and MgATP2- in the reverse direction. 2. Primary reciprocal plots rule out the possibility of a Ping Pong mechanism for both forms of the enzyme. 3. Analysis of the kinetic data by an appropriate computer program gave the following Km values for the type 1 enzyme: AMP, 0.33mm±0.1; MgATP2-, 0.95mm±0.13; ADP, 0.12mm±0.03; MgADP-, 0.22mm±0.04. Values for the type 2 enzyme were: AMP, 0.27mm±0.03; MgATP2-, 0.40mm±0.05; ADP, 0.08mm±0.07; MgADP-, 0.20mm±0.04. 4. Product inhibition studies were done by studying the reverse reaction. With ADP as product inhibitor competitive inhibition patterns were obtained with AMP and/or MgATP2- as variable substrate. Similar results were obtained for product inhibition by MgADP- with AMP as variable substrate. The results are consistent with a Rapid Equilibrium Random mechanism. 5. Secondary plots of slope versus product concentration were linear. The data were fitted to the appropriate equation and analysed by computer to give values for the product inhibition constants. 6. Differences between the values of certain kinetic constants for the two forms of the enzyme were observed.

MECHANISM OF Fe++-CYTOCROME c REDUCTASE OF FERROBACILLUS FERROOXIDANS

1967 ◽  
Vol 45 (10) ◽  
pp. 1547-1556 ◽  
Author(s):  
George A. Din ◽  
Isamu Suzuki
Keyword(s):  
Cytochrome C ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Heme Iron ◽  
Non Heme Iron ◽  
Oxidation Reduction ◽  
Dead End ◽  
Ping Pong ◽  
Stable Forms ◽  
Variable Substrate

The mechanism of Fe++-cytochrome c reductase was investigated. Kinetic studies on initial velocity and product inhibition, as well as spectrofluorometric studies, were consistent with a Ping Pong Bi Bi mechanism with two stable forms of enzyme. The Km values for the substrates were found to be 0.59 mM for Fe++ and 0.085 mM for cytochrome c. The inhibition constants for Fe+++ and reduced cytochrome c were 0.137 mM and 0.0135 mM, respectively. The oxidation–reduction of non-heme iron bound to the enzyme protein was implicated as the responsible factor for the oscillation of the enzyme between its two forms. NaCl was a dead-end inhibitor binding the ferrous form of the enzyme, showing an uncompetitive inhibition with Fe++ as a variable substrate.

Adenylate Kinase from Maize Leaves: True Substrates, Inhibition by P1 , P5-di(adenosine-5′) pentaphosphate and Kinetic Mechanism

1990 ◽  
Vol 45 (6) ◽  
pp. 607-613 ◽  
Author(s):  
Leszek A. Kleczkowski ◽  
Douglas D. Randall ◽  
Warren L. Zahler
Keyword(s):  
Adenylate Kinase ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Reverse Reaction ◽  
Plant Tissues ◽  
Forward Reaction ◽  
Higher Plant ◽  
Maize Leaves ◽  
Variable Substrate ◽  
Free Magnesium

Abstract Purified maize leaf adenylate kinase (AK) was shown to use one molecule each of free ADP and Mg-ADP as well as free AM P and Mg-ATP as substrates in the forward and reverse reaction, respectively. This was deduced from substrate kinetic studies which were carried out under conditions of strictly defined concentrations of free and Mg-complexed adenylate species and under controlled free magnesium levels. Apparent Km values of the substrates of AK were 3 and 6 μM for ADP and Mg-ADP, respectively (forward reaction), and 69 and 25 μM for free AMP and Mg-ATP, respectively (reverse reaction). The enzyme was competitively inhibited by P1,P5-di(adenosine-5′)pentaphosphate (Ap5A), a bisubstrate analog of AK reaction, with apparent Ki values in the range of 11 -80 nM , depending on variable substrate. Substrate kinetic studies and inhibition patterns with Ap5A suggested a sequential random kinetic mechanism in both directions of the reaction. These properties of a higher plant AK are similar or analogous to those previously established for the enzyme from yeast and non-plant tissues.

scholarly journals Purification and characterization of morphinone reductase from Pseudomonas putida M10

1994 ◽  
Vol 301 (1) ◽  
pp. 97-103 ◽  
Author(s):  
C E French ◽  
N C Bruce
Keyword(s):  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Apparent Homogeneity ◽  
Ping Pong

The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.

Studies on the kinetics and regulation of glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 439-450 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Forward Direction ◽  
Product Inhibition ◽  
Reverse Direction ◽  
High Concentrations ◽  
Glutamate Synthesis ◽  
Inhibition Studies

A 190-fold purified preparation of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium was used for the determination of kinetic parameters of the substrates, NADPH, NH4+, and α-ketoglutarate, in the direction of glutamate synthesis, and NADP+ and glutamate in the reverse direction. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme. Based on an analysis of the data derived from initial velocity and product inhibition studies, the reaction mechanism was postulated to be ordered Ter Bi with NADPH as the first substrate to bind in the forward direction, and NADP+ binding first in the reverse direction.Of the several metabolites tested for a possible function in the regulation of GDH activity, only L-malate and L-glutamine appeared to exert an appreciable influence on the enzyme. ATP and AMP at a concentration of 0.8 mM were found to enhance GDH activity by 68% and 6%, respectively, but at high concentrations, both the adenine nucleotides proved to be inhibitory.

Glutamate Dehydrogenase from Medicago sativa L., Purification and Comparative Kinetic Studies of the Organ-Specific Multiple Forms

1980 ◽  
Vol 35 (5-6) ◽  
pp. 406-415 ◽  
Author(s):  
Marianne Nagel ◽  
Hartmann
Keyword(s):  
Medicago Sativa ◽  
Glutamate Dehydrogenase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Enzyme Preparations ◽  
Organ Specific ◽  
Nad Oxidoreductase ◽  
Medicago Sativa L ◽  
Inhibition Studies

Abstract NAD-specific glutamate dehydrogenase [L-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-pattems of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measure­ ments and product inhibition studies are consistent with an ordered ternary-binary kinetic mecha­ nism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.

Kinetic Studies of Phosphoribosyl-formylglycineamidine Synthetase

1972 ◽  
Vol 50 (5) ◽  
pp. 490-500 ◽  
Author(s):  
Samuel Y. Chu ◽  
J. Frank Henderson
Keyword(s):  
Ammonium Chloride ◽  
Initial Velocity ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Ping Pong ◽  
Inhibition Constants ◽  
Inhibition Studies

Initial velocity and product inhibition studies of phosphoribosyl-formylglycineamidine synthetase indicate that the reaction involves a fully ping pong mechanism in which glutamine binds to the free enzyme and glutamate is released before the addition of ATP. ADP is released, and phosphoribosyl-formylglycineamide then binds; the liberation of Pi is rapid, and phosphoribosyl-formylglycineamidine is the last product released from the enzyme. The Km values for glutamine, ATP, and phosphoribosyl-formylglycineamide are 1.1 × 10−4 M, 1.5 × 10−3 M, and 1.1 × 10−4 M, respectively. The Km value for ammonium chloride is 7.5 × 10−3 M, and the ratio of Vmax values with ammonium chloride and glutamine is 1/40. The inhibition constants for FGAM and Pi were calculated to be 1.3 × 10−4 M and 6.45 × 10−3 M, respectively.

scholarly journals A product-inhibition study of bovine liver glutamate dehydrogenase

1975 ◽  
Vol 151 (2) ◽  
pp. 305-318 ◽  
Author(s):  
P C Engel ◽  
S S Chen
Keyword(s):  
Glutamate Dehydrogenase ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Random Order ◽  
Bovine Liver ◽  
Product Inhibition ◽  
Rate Equations ◽  
Sodium Phosphate Buffer ◽  
Rapid Equilibrium ◽  
Variable Substrate

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.

scholarly journals The kinetic properties of human placental choline acetyltransferase

1971 ◽  
Vol 125 (3) ◽  
pp. 857-863 ◽  
Author(s):  
D. Morris ◽  
A. Maneckjee ◽  
Catherine Hebb
Keyword(s):  
Sodium Chloride ◽  
Choline Acetyltransferase ◽  
Product Inhibition ◽  
Rabbit Brain ◽  
Kinetic Properties ◽  
Forward Reaction ◽  
Acetyl Coa ◽  
No Inhibition ◽  
Ping Pong ◽  
Michaelis Constants

1. Michaelis constants for human placental choline acetyltransferase were shown to be dependent on the concentration of the second substrate present. The primary plots indicate a sequential rather than a Ping Pong mechanism and are of the same type with 300mm- and 500mm-sodium chloride. 2. Similar results have been obtained with rabbit brain choline acetyltransferase. 3. Product inhibition of the forward reaction has been studied. CoA inhibits competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibits competitively with respect to choline and non-competitively with respect to acetyl-CoA. No inhibition is given by acetylcholine when the enzyme is saturated with choline. 4. It is concluded that human placental choline acetyltransferase has an ordered mechanism of the Theorell–Chance type.

Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

2020 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
Ravneet K. Grewal ◽  
Baldeep Kaur ◽  
Gagandeep Kaur
Keyword(s):  
Metal Ions ◽  
Competitive Inhibition ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Cost Effective ◽  
Starch Hydrolysis ◽  
Divalent Metal Ions ◽  
Activity Data ◽  
Time Saving ◽  
Alkaline Conditions

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

scholarly journals Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Juan Carlos Serrani-Yarce ◽  
Luis Escamilla-Trevino ◽  
Jaime Barros ◽  
Lina Gallego-Giraldo ◽  
Yunqiao Pu ◽  
...  
Keyword(s):  
Negative Impact ◽  
Lignin Content ◽  
Brachypodium Distachyon ◽  
Lignin Biosynthesis ◽  
Reverse Direction ◽  
Wall Structure ◽  
Kinetic Properties ◽  
Loss Of Function ◽  
Down Regulation ◽  
Lignin Modification

Abstract Background Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called “esters” pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the “reverse” direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an “incomplete” shikimate shunt. Results The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. Conclusions Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

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