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.

Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin-containg carboxylase

1969 ◽  
Vol 47 (7) ◽  
pp. 697-710 ◽  
Author(s):  
Helen A. Feir ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Pyruvate Carboxylase ◽  
Active Sites ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Metal Cofactor ◽  
Michaelis Constants

Pyruvate carboxylase was partially purified from Aspergillus niger and the properties were studied. The enzyme was found to be cold-labile and protected by 25% glycerol. The pH optimum was determined to be 7.9–8.0. The enzyme was shown to be a biotin-containing enzyme by its inactivation by avidin and protection against such inactivation by biotin. The enzyme activity was stimulated by K+ ions and inhibited by Na+ ions. Acetyl-CoA had no effect on enzyme activity, but L-aspartate was inhibitory. Apparent Michaelis constants were determined for the substrates and metal cofactor involved, i.e. pyruvate, ATP, bicarbonate, and Mg2+.Initial-velocity studies were carried out at varied concentrations of substrates in order to determine the true Michaelis constants and to elucidate the kinetic mechanism of reaction. Product-inhibition studies were carried out with each product (ADP, Pi, and oxalacetate) in combination with every substrate (ATP, bicarbonate, and pyruvate). From these kinetic studies and the existing knowledge on biotin-containing carboxylases, a mechanism was proposed for the action of pyruvate carboxylase which involves three independently active sites on the enzyme, one for each substrate. The interactions between the sites were visualized as being mediated by carboxybiotin formed on the enzyme. A steady-state rate equation was derived that satisfied kinetic results obtained.

scholarly journals Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Juan L. Rendón ◽  
Mauricio Miranda-Leyva ◽  
Alberto Guevara-Flores ◽  
José de Jesús Martínez-González ◽  
Irene Patricia del Arenal ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Active Form ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Hysteretic Behavior ◽  
Low Concentrations ◽  
Mechanistic Basis ◽  
High Concentrations ◽  
Thioredoxin Glutathione Reductase

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii

1991 ◽  
Vol 69 (4) ◽  
pp. 223-231 ◽  
Author(s):  
Mamdouh Y. Kamel ◽  
Afaf S. Fahmy ◽  
Abdel H. Ghazy ◽  
Magda A. Mohamed
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Catalytic Mechanism ◽  
Kinetic Studies ◽  
Purine Nucleoside ◽  
Hyalomma Dromedarii ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Apparent Homogeneity ◽  
Camel Tick

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56 000 – 58 000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of β-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6–7 and 8–9. The enzyme was completely inactivated by thiol reagents and reactivated by excess β-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.Key words: Acarina, Hyalomma dromedarii, purine nucleoside phosphorylase, kinetics, active site, catalytic mechanism.

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 Kinetic studies of formate dehydrogenase

1970 ◽  
Vol 120 (4) ◽  
pp. 763-769 ◽  
Author(s):  
D. Peacock ◽  
D. Boulter
Keyword(s):  
Carbon Dioxide ◽  
Formate Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Reverse Reaction ◽  
Enzyme Form ◽  
Rate Limiting Step ◽  
Measurable Rate ◽  
Rate Limiting

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD+ binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme–coenzyme complexes. 4. The secondary plots of the inhibitions with HCO3− and NO3− are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the Km values for NAD+ and NADH.

scholarly journals Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

2000 ◽  
Vol 345 (3) ◽  
pp. 687-692 ◽  
Author(s):  
Edward H. WALKER ◽  
Christopher E. FRENCH ◽  
Deborah A. RATHBONE ◽  
Neil C. BRUCE
Keyword(s):  
Aldose Reductase ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Site Directed Mutagenesis ◽  
Assay Method ◽  
Sequence Comparisons ◽  
Biotransformation Process ◽  
Steady State Kinetic ◽  
Catalytic Role

Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP+-dependent oxidation of morphine and codeine to morphinone and codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.

scholarly journals Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

2008 ◽  
Vol 191 (5) ◽  
pp. 1472-1479 ◽  
Author(s):  
Jinki Yeom ◽  
Che Ok Jeon ◽  
Eugene L. Madsen ◽  
Woojun Park
Keyword(s):  
Growth Rate ◽  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Ferric Reductase ◽  
Wild Type ◽  
Cell Extracts ◽  
Mutant Strains ◽  
First Time

ABSTRACT Pseudomonas putida harbors two ferredoxin-NADP+ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k cat/Km value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.

Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase

2001 ◽  
Vol 357 (1) ◽  
pp. 283-288 ◽  
Author(s):  
Donald A. VESSEY ◽  
Michael KELLEY
Keyword(s):  
Reaction Mechanism ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Product Inhibition ◽  
Bivalent Cation ◽  
Medium Chain ◽  
Apparent Homogeneity ◽  
Ping Pong ◽  
Inhibition Studies

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg2+ results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg2+ and ATP are varied together.

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 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.

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