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 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.

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.

Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe

1998 ◽  
Vol 76 (4) ◽  
pp. 637-644 ◽  
Author(s):  
C Stan Tsai ◽  
Q Chen
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Pentose Phosphate ◽  
Oxidative Decarboxylation ◽  
Divalent Metal Ions ◽  
Basic Residue ◽  
Phosphogluconate Dehydrogenase ◽  
Steady State Kinetic

6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe. The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+. Steady-state kinetic studies were undertaken. Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP+-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2. Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.Key words: yeast, 6-phosphogluconate dehydrogenase.

scholarly journals Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase

1977 ◽  
Vol 167 (3) ◽  
pp. 787-798 ◽  
Author(s):  
Roy D. Waight ◽  
Paul Leff ◽  
William G. Bardsley
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Rate Equation ◽  
Substrate Concentration ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Flip Flop ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Recent Classification

1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis–Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/ν) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis–Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.

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.

The roles of Tyr91 and Lys162 in general acid–base catalysis in the pigeon NADP+-dependent malic enzyme

2008 ◽  
Vol 411 (3) ◽  
pp. 467-473 ◽  
Author(s):  
Cheng-Chin Kuo ◽  
Kuan-Yu Lin ◽  
Yau-Jung Hsu ◽  
Shu-Yu Lin ◽  
Yu-Tsen Lin ◽  
...  
Keyword(s):  
Malic Enzyme ◽  
Kinetic Studies ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Decarboxylase Activity ◽  
Wild Type ◽  
Acid Base ◽  
Enzymatic Mechanism ◽  
General Acid ◽  
Steady State Kinetic

The role of general acid–base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr91 and Lys162 residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in kcat values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the kcat values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(kcat) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pKa value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys162 as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr91 residue could form a hydrogen bond with Lys162 to act as a catalytic dyad that contributes a proton to complete the enol–keto tautomerization.

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.

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