Kinetic mechanism of phosphorylase a, II. Isotope exchange studies at equilibrium

1970 ◽  
Vol 48 (7) ◽  
pp. 755-758 ◽  
Author(s):  
H. D. Engers ◽  
W. A. Bridger ◽  
N. B. Madsen
Keyword(s):  
Exchange Rates ◽  
Isotope Exchange ◽  
Initial Rate ◽  
Kinetic Mechanism ◽  
Rabbit Muscle ◽  
Glucose Inhibition ◽  
Rate Limiting Step ◽  
Equilibrium Exchange ◽  
Muscle Phosphorylase ◽  
Rate Limiting

In order to confirm the kinetic mechanism which was proposed for rabbit muscle phosphorylase a on the basis of initial rate studies and UDP-glucose inhibition experiments, isotope exchange studies at equilibrium were performed, both in the presence and absence of the modifier AMP.Both the 14C-glucose [Formula: see text] and the [Formula: see text]1-phosphate equilibrium exchange rates increased to a maximum as the concentrations of the varied substrates became saturating, either in the presence or absence of AMP. The plateaus observed in these experiments indicate the lack of inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised, and confirms the proposed random addition of substrates to the enzyme.The fact that similar exchange rates were observed for either reaction direction reinforced the concept that rapid equilibrium conditions apply to the phosphorylase a mechanism; i.e. the interconversion of the ternary complexes tends to be the rate-limiting step in the reaction sequence.Maximal velocities determined from initial rate data reported in the previous paper agreed with those calculated from isotope exchange rates.

Kinetic mechanism of phosphorylase a. I. Initial velocity studies

1970 ◽  
Vol 48 (7) ◽  
pp. 746-754 ◽  
Author(s):  
H. D. Engers ◽  
S. Shechosky ◽  
N. B. Madsen
Keyword(s):  
Dissociation Constants ◽  
Glycogen Metabolism ◽  
Kinetic Mechanism ◽  
Rabbit Muscle ◽  
Reaction Sequence ◽  
Rate Limiting Step ◽  
Muscle Phosphorylase ◽  
Inhibition Studies ◽  
Rate Limiting ◽  
Reaction Direction

Initial rate studies on rabbit muscle phosphorylase a were carried out in order to assign a kinetic mechanism to this enzyme, which plays an important role in the control of glycogen metabolism. Initial velocities were measured with varied concentrations of both substrates in each reaction direction, both in the presence and the absence of the modifier AMP. Data were analyzed with double-reciprocal plots and secondary replots of slopes and intercepts to yield kinetically derived dissociation constants which may be compared with dissociation constants determined by independent methods. Inhibition studies using UDP-glucose as a substrate analogue are also reported. Inhibition is competitive with glucose 1-phosphate and P1 and noncompetitive with glycogen.A suitable rate equation for this system has been derived, and it should apply to other two-substrate enzyme–modifier systems exhibiting similar kinetics. The results of this detailed kinetic analysis, in conjunction with the isotope exchange studies at equilibrium which are reported in the following paper, suggest the kinetic mechanism of phosphorylase a to be rapid equilibrium random Bi Bi, i.e. random addition of substrates to the enzyme, with ternary complex interconversion as the rate-limiting step in the reaction sequence.

scholarly journals Substrate specificity of sheep liver sorbitol dehydrogenase

1998 ◽  
Vol 330 (1) ◽  
pp. 479-487 ◽  
Author(s):  
I. Rune LINDSTAD ◽  
Peter KÖLL ◽  
John S. McKINLEY-McKEE
Keyword(s):  
Substrate Specificity ◽  
Ternary Complex ◽  
Kinetic Mechanism ◽  
Sorbitol Dehydrogenase ◽  
Hydroxyl Group ◽  
Enzyme Active Site ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

scholarly journals Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

1989 ◽  
Vol 261 (3) ◽  
pp. 935-943 ◽  
Author(s):  
C Forte-McRobbie ◽  
R Pietruszko
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Maximal Velocity ◽  
Semialdehyde Dehydrogenase ◽  
Rate Limiting Step ◽  
Dead End ◽  
Inhibition Studies ◽  
Rate Limiting ◽  
Competitive Pattern ◽  
Pattern Versus

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.

scholarly journals Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

1972 ◽  
Vol 130 (2) ◽  
pp. 397-410 ◽  
Author(s):  
H. G. Britton ◽  
J. B. Clarke
Keyword(s):  
Chemical Equilibrium ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Kinetic Measurements ◽  
Rate Limiting Step ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Intramolecular Transfer

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

Succinyl Coenzyme A Synthetase of Escherichia coli: Initial Rate Kinetics of Succinyl-CoA Cleavage and Isotope Exchange Studies

1973 ◽  
Vol 51 (1) ◽  
pp. 44-55 ◽  
Author(s):  
Frank J. Moffet ◽  
W. A. Bridger
Keyword(s):  
Isotope Exchange ◽  
Coenzyme A ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Constant Ratio ◽  
Rate Kinetics ◽  
Kinetics Of ◽  
Succinyl Coenzyme A Synthetase

Initial rate kinetic studies of succinyl coenzyme A synthetase of E. coli in the direction of succinyl-CoA cleavage are consistent with the operation of a partially random sequential kinetic mechanism with initial binding of ADP followed by random association of succinyl-CoA and Pi. The mechanism is analogous to that proposed previously for the succinyl-CoA formation reaction, and thus the kinetic mechanism of the overall reversible succinyl-CoA synthetase reaction appears to be symmetrical.Studies of the kinetics of [Formula: see text] isotope exchange at equilibrium show that this partially random sequential kinetic mechanism is not an exclusive pathway. [Formula: see text] isotope exchange rates did not show complete substrate inhibition when CoA or succinate was varied in constant ratio with Pi. However, when CoA or succinate was varied in constant ratio with succinyl-CoA, nearly complete substrate inhibition was observed. These results can be interpreted in terms of a wide variety of minor pathways of substrate binding and product release available to the enzyme under various conditions.

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 Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

2006 ◽  
Vol 399 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Zhen Shi ◽  
Gloria C. Ferreira
Keyword(s):  
Protoporphyrin Ix ◽  
Kinetic Mechanism ◽  
Wild Type ◽  
Rate Limiting Step ◽  
Transition State Analogue ◽  
Mammalian Cell Cultures ◽  
Kinetic Mechanisms ◽  
Specificity Constant ◽  
Order Of Magnitude ◽  
Rate Limiting

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248–257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (Kiapp values of 1 μM and 2.3 μM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/Km, protoporhyrin IX) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.

A Kinetic Study on the Methanogenesis Process in Anaerobic Digestion

1989 ◽  
Vol 21 (4-5) ◽  
pp. 175-186 ◽  
Author(s):  
C. Y. Lin ◽  
T. Noike ◽  
H. Furumai ◽  
J. Matsumoto
Keyword(s):  
Anaerobic Digestion ◽  
Substrate Concentration ◽  
Volatile Fatty Acid ◽  
Kinetic Mechanism ◽  
High Concentration ◽  
Two Phase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
The Relationship ◽  
Rate Limiting

Results obtained from experiments on two-phase anaerobic digestion using a high concentration of a volatile fatty acid (VFA) mixture were used to elucidate the kinetic mechanism of the methanogenesis process. The mixture consisted of the major intermediate products of anaerobic digestion, i.e., acetic acid (HAc), propionic acid (HPr) and butyric acid (HBu). The relationship between the rate of substrate utilization and substrate concentration in the digesters was in the form of a Michaelis-Menten equation. The rate-limiting step of the methanogenesis process, i.e., the conversion of HAc to methane, was speeded up in the digesters and this was proved kinetically. A method for determining kinetic constants for substrate-specific microorganisms was suggested. A simulation model for predicting the effluent substrate concentration was demonstrated. The effluent substrate concentration of an anaerobic digester fed by a multisubstrate was found to be simulatively predictable from its influent component substrates.

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