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.

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.

Dioxygen Activation at Non-Heme Iron: Insights from Rapid Kinetic Studies

2007 ◽  
Vol 40 (7) ◽  
pp. 510-521 ◽  
Author(s):  
Ivan V. Korendovych ◽  
Sergey V. Kryatov ◽  
Elena V. Rybak-Akimova
Keyword(s):  
Kinetic Studies ◽  
Heme Iron ◽  
Dioxygen Activation ◽  
Non Heme Iron

scholarly journals Kinetic studies of rat liver hexokinase D (glucokinase) in non-co-operative conditions show an ordered mechanism with MgADP as the last product to be released

2003 ◽  
Vol 371 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Octavio MONASTERIO ◽  
María Luz CÁRDENAS
Keyword(s):  
Rat Liver ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Nitrobenzoic Acid ◽  
Binary Complexes ◽  
Dead End ◽  
Straight Line ◽  
Inactivation Constant ◽  
Reciprocal Value

The kinetic mechanism of rat liver hexokinase D ('glucokinase') was studied under non-co-operative conditions with 2-deoxyglucose as substrate, chosen to avoid uncertainties derived from the co-operativity observed with the physiological substrate, glucose. The enzyme shows hyperbolic kinetics with respect to both 2-deoxyglucose and MgATP2-, and the reaction follows a ternary-complex mechanism with Km = 19.2±2.3mM for 2-deoxyglucose and 0.56±0.05mM for MgATP2-. Product inhibition by MgADP- was mixed with respect to MgATP2- and was largely competitive with respect to 2-deoxyglucose, suggesting an ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. Dead-end inhibition by N-acetylglucosamine, AMP and the inert complex CrATP [the complex of ATP with chromium in the 3+ oxidation state, i.e. Cr(III)—ATP], studied with respect to both substrates, also supports an ordered mechanism with 2-deoxyglucose as first substrate. AMP appears to bind both to the free enzyme and to the E·dGlc complex. Experiments involving protection against inactivation by 5,5′-dithiobis-(2-nitrobenzoic acid) support the existence of the E·MgADP- and E·AMP complexes suggested by the kinetic studies. MgADP-, AMP, 2-deoxyglucose, glucose and mannose were strong protectors, supporting the existence of binary complexes with the enzyme. Glucose 6-phosphate failed to protect, even at concentrations as high as 100mM, and MgATP2- protected only slightly (12%). The inactivation results support the postulated ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. In addition, the straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugar-binding site in hexokinase D.

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.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. I. Survey of enzymes and properties of sulfite : cytochrome c oxidoreductase

1970 ◽  
Vol 48 (3) ◽  
pp. 334-343 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Marked Effect ◽  
Sulfite Oxidase ◽  
Heme Iron ◽  
Free Enzyme ◽  
Reaction Velocity ◽  
Thiobacillus Thioparus ◽  
Non Heme Iron

Enzymes concerned with the oxidation of thiosulfate were investigated in extracts of Thiobacillus thioparus. The organism possessed sulfite oxidase as well as adenosine-5′-phosphosulfate reductase and thiosulfate-oxidizing enzyme. Sulfite oxidase was purified 160-fold and the properties were studied. The enzyme had a molecular weight of 54 000 and one non-heme iron. The pH had a marked effect on reaction velocity and Km for sulfite, and the pK values for free enzyme and enzyme–sulfite complex were determined as 8.9 and 6.2, respectively. Chloride inhibition was noncompetitive and phosphate was uncompetitive with respect to sulfite. In many properties the T. thioparus enzyme was similar to the enzyme isolated from Thiobacillus novellus.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. III. Properties of thiosulfate-oxidizing enzyme and proposed pathway of thiosulfate oxidation

1970 ◽  
Vol 48 (3) ◽  
pp. 355-363 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Heme Iron ◽  
Time Dependent ◽  
Strong Inhibitor ◽  
Thiobacillus Thioparus ◽  
Ph Response ◽  
Thiosulfate Oxidation ◽  
Non Heme Iron ◽  
Thiobacillus Neapolitanus

Thiosulfate-oxidizing enzyme was purified from Thiobacillus thioparus extracts 120- to 160-fold and the properties were studied. The enzyme had a molecular weight of 115 000 and contained 2 moles of non-heme iron. Ferricyanide was a much better electron acceptor than cytochrome c, but with cytochrome c the Km for thiosulfate was lowered from 0.1 mM to 5 μM and the pH response of the enzyme changed. Sulfite was a very strong inhibitor destroying 50% of the activity at 5 μM. The inhibition was time-dependent and essentially irreversible. Properties of the T. thioparus enzyme were compared to those of thiosulfate-oxidizing enzyme isolated from Thiobacillus neapolitanus and Ferrobacillus ferrooxidans. A pathway of thiosulfate oxidation is proposed, and metabolic roles of various enzymes studied in T. thioparus are discussed.

Split-soret cytochrome c from desulfovibrio desulfuricans ATCC 27774, a novel protein that contains both heme and non-heme iron centers

1997 ◽  
Vol 67 (1-4) ◽  
pp. 111
Author(s):  
Cristina Costa ◽  
B. Devreese ◽  
J. Van Beeumen ◽  
V. Papaefthymiou ◽  
A. Simopoulos ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Desulfovibrio Desulfuricans ◽  
Heme Iron ◽  
Non Heme Iron ◽  
Novel Protein

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.

Kinetic studies of sulfite; cytochrome c oxidoreductase, thiosulfate-oxidizing enzyme, and adenostne-5′-phosphosulfate reductase from Thiobacillus thioparus

1970 ◽  
Vol 48 (5) ◽  
pp. 594-603 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Cytochrome C ◽  
Initial Velocity ◽  
Enzyme Reaction ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Thiobacillus Thioparus ◽  
Inhibition Studies

Kinetic studies were carried out on three enzymes purified from Thiobacillus thioparus: sulfite: cytochrome c oxidoreductase, thiosulfate-oxidizing enzyme, and adenosine-5′-phosphosulfate reductase. From the initial velocity and product inhibition studies a tentative kinetic mechanism was proposed for each enzyme reaction.

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