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 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 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.

Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe

1998 ◽  
Vol 76 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C Stan Tsai ◽  
Q Chen
Keyword(s):  
Pentose Phosphate Pathway ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Phosphoryl Group ◽  
Maximal Velocity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Yeast Hexokinase

Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pombe have been purified 250-fold by an identical three-step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S. pombe (fission yeast). Hexokinase promotes Mg2+-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose. The Mg2+activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group. G6PDH catalyzes NADP+-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+as the leading reactant. High NADP+concentration inhibits the dehydrogenase by forming the dead-end ternary complex. In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.Key words: yeast hexokinase, glucose-6-phosphate dehydrogenase.

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 Emergence of protocellular growth laws

2007 ◽  
Vol 362 (1486) ◽  
pp. 1841-1845 ◽  
Author(s):  
Tristan Rocheleau ◽  
Steen Rasmussen ◽  
Peter E Nielsen ◽  
Martin N Jacobi ◽  
Hans Ziock
Keyword(s):  
Kinetic Studies ◽  
Product Inhibition ◽  
Replication Process ◽  
Gene Replication ◽  
Math Biol ◽  
Curr Opin Cell Biol ◽  
Growth Laws ◽  
Internal Gene ◽  
The Many ◽  
The Individual

Template-directed replication is known to obey a parabolic growth law due to product inhibition (Sievers & Von Kiedrowski 1994 Nature 369 , 221; Lee et al . 1996 Nature 382 , 525; Varga & Szathmáry 1997 Bull. Math. Biol . 59 , 1145). We investigate a template-directed replication with a coupled template catalysed lipid aggregate production as a model of a minimal protocell and show analytically that the autocatalytic template–container feedback ensures balanced exponential replication kinetics; both the genes and the container grow exponentially with the same exponent. The parabolic gene replication does not limit the protocellular growth, and a detailed stoichiometric control of the individual protocell components is not necessary to ensure a balanced gene–container growth as conjectured by various authors (Gánti 2004 Chemoton theory ). Our analysis also suggests that the exponential growth of most modern biological systems emerges from the inherent spatial quality of the container replication process as we show analytically how the internal gene and metabolic kinetics determine the cell population's generation time and not the growth law (Burdett & Kirkwood 1983 J. Theor. Biol . 103 , 11–20; Novak et al . 1998 Biophys. Chem . 72 , 185–200; Tyson et al . 2003 Curr. Opin. Cell Biol . 15 , 221–231). Previous extensive replication reaction kinetic studies have mainly focused on template replication and have not included a coupling to metabolic container dynamics (Stadler et al . 2000 Bull. Math. Biol . 62 , 1061–1086; Stadler & Stadler 2003 Adv. Comp. Syst . 6 , 47). The reported results extend these investigations. Finally, the coordinated exponential gene–container growth law stemming from catalysis is an encouraging circumstance for the many experimental groups currently engaged in assembling self-replicating minimal artificial cells (Szostak 2001 et al . Nature 409 , 387–390; Pohorille & Deamer 2002 Trends Biotech . 20 123–128; Rasmussen et al . 2004 Science 303 , 963–965; Szathmáry 2005 Nature 433 , 469–470; Luisi et al . 2006 Naturwissenschaften 93 , 1–13). 1

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