scholarly journals The kinetics of interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide–adenine dinucleotide and lactate

1973 ◽  
Vol 135 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Nigel G. Bennett ◽  
Herbert Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Dead Time ◽  
Muscle Lactate ◽  
Rapid Formation ◽  
Steady State Rate ◽  
Pig Muscle ◽  
State Rate ◽  
Enzyme Complexes ◽  
Kinetics Of

Oxamate competes with pyruvate for the substrate binding site on the ENADH complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD+ and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of ENADHOxamate, the rate of formation of ENADH could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form ENADHPyruvate.

scholarly journals The resolution of some steps of the reactions of lactate dehydrogenase with its substrates

1968 ◽  
Vol 108 (5) ◽  
pp. 793-796 ◽  
Author(s):  
H. D'A. Heck ◽  
C. H. McMurray ◽  
H. Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Ternary Complex ◽  
Spectrophotometric Methods ◽  
Steady State Rate ◽  
State Rate

1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD+ and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD+ per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme–NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided.

scholarly journals Effect of pyrophosphate ions and alkaline pH on the kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase

1991 ◽  
Vol 273 (3) ◽  
pp. 691-693 ◽  
Author(s):  
J P Hill ◽  
P D Buckley ◽  
L F Blackwell ◽  
R L Motion
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Alkaline Ph ◽  
Activation Effect ◽  
Ph Values ◽  
Sheep Liver ◽  
Steady State Rate ◽  
State Rate ◽  
Rate Limiting ◽  
Kinetics Of

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

On the mechanism of NADP + -linked isocitrate dehydrogenase from heart mitochondria. I. The kinetics of dissociation of NADPH from its enzyme complex

1982 ◽  
Vol 214 (1196) ◽  
pp. 369-387 ◽  
Keyword(s):  
Steady State ◽  
Isocitrate Dehydrogenase ◽  
Oxidative Decarboxylation ◽  
Dissociation Kinetics ◽  
Fluorescence Measurements ◽  
Rate Limiting Step ◽  
Ligand Displacement ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

The kinetics of dissociation of NADPH from its complex with isocitrate dehydrogenase, and from the abortive complex of enzyme, Mg 2+ , isocitrate and NADPH, have been studied in phosphate and triethanolamine buffers by means of rapid fluorescence measurements. The reactions are complex, and it is suggested that a conformational equilibrium of each of the complexes is involved, and that this conformational change is also responsible for a slow approach to the steady-state rate of oxidative decarboxylation observed previously in triethanolamine buffer under certain conditions (K. Dalziel, N. McFerran, B. Matthews & C. H. Reynolds, Biochem . J . 171, 743‒750 (1978)). It is concluded that release of free NADPH product is not the rate-limiting step in oxidative decarboxylation in the steady state. The validity of the ligand displacement method used to measure the dissociation kinetics of the enzyme‒NADPH complex has been studied by computer simulation.

A New Method for Determining Individual Rate Constants for Specific Non-chromophoric Substrates of Proteolytic Enzymes

1975 ◽  
Vol 53 (4) ◽  
pp. 564-571
Author(s):  
Lewis J. Brubacher
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Proteolytic Enzymes ◽  
Enzyme Mechanism ◽  
Steady State Kinetics ◽  
Steady State Rate ◽  
State Rate ◽  
Individual Rate ◽  
Kinetics Of ◽  
Hydrolysis Of

Equations are developed for the pre-steady state kinetics of the proteolytic enzyme-catalyzed hydrolysis of a substrate A in the presence of a monitoring substrate (or covalent inhibitor) S of known properties. A two-intermediate acyl–enzyme mechanism is assumed in which the first intermediate is in instantaneous equilibrium with enzyme and substrate. The appearance of the first product of substrate S is characterized by two relaxation rate constants. From these constants it is possible to determine the dissociation constant and the acylation and deacylation rate constants of substrate A. Criteria are also developed for using the steady state rate parameters of A to establish conditions for which the slower relaxation process is equivalent to the deacylation rate constant of A. The technique of premixing enzyme with substrate A has certain advantages in this approach.

Effects of modifiers on the kinetics of two-substrate enzyme reactions

1968 ◽  
Vol 46 (11) ◽  
pp. 1381-1396 ◽  
Author(s):  
J. Frank Henderson
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Rate Equations ◽  
Enzyme Reactions ◽  
Ping Pong ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

Steady state rate equations have been derived for ordered bi bi and ping pong bi bi reactions in which there are (a) one or two nonsubstrate modifiers, (b) two different binding sites for a single nonsubstrate modifier, (c) one or two substrates acting as modifiers, and (d) both nonsubstrate modifiers and substrates acting as modifiers. The deviation of these equations from the Michaelis–Menten equation is shown and methods are suggested by which many of these mechanisms can be distinguished experimentally.

scholarly journals Transient-kinetic studies of pig muscle lactate dehydrogenase

1971 ◽  
Vol 121 (2) ◽  
pp. 235-240 ◽  
Author(s):  
R. A. Stinson ◽  
H. Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Hydride Transfer ◽  
Kinetic Studies ◽  
Muscle Lactate ◽  
First Order ◽  
Ph Values ◽  
Liver Alcohol Dehydrogenase ◽  
Pig Muscle ◽  
Rate Limiting

1. The very fast pre-steady-state formation of NADH catalysed by pig M4 lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s−1) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s−1). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme–NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M4 isoenzyme were drawn from a comparison of these results with those obtained with the H4 isoenzyme and liver alcohol dehydrogenase.

A COMPARISON BETWEEN THE INITIAL RATE EXPRESSIONS OBTAINED UNDER STRICT CONDITIONS AND THE RAPID EQUILIBRIUM ASSUMPTION USING, AS EXAMPLE, A FOUR SUBSTRATE ENZYME REACTION

2011 ◽  
Vol 10 (05) ◽  
pp. 659-678
Author(s):  
J. M. YAGO ◽  
C. GARRIDO-DEL SOLO ◽  
M. GARCIA-MORENO ◽  
R. VARON ◽  
F. GARCIA-SEVILLA ◽  
...  
Keyword(s):  
Steady State ◽  
Rate Equation ◽  
Initial Rate ◽  
Enzyme Reaction ◽  
Rate Equations ◽  
Steady State Rate ◽  
State Rate ◽  
Rapid Equilibrium ◽  
Kinetics Of ◽  
Substrate Concentrations

The software WinStes, developed by our group, is used to derive the strict steady-state initial rate equation of the reaction mechanism of CTP:sn-glycerol-3-phosphate cytidylyltransferase [EC 2.7.7.39] from Bacillus subtilis. This enzyme catalyzes a reaction with two substrates and operates by a random ordered binding mechanism with two molecules of each substrate. The accuracy of the steady-state rate equation derived is checked by comparing the rate values it provides with those obtained from the simulated progress curves. To analyze the kinetics of this enzyme using the strict steady-state initial rate equation, several curves for different substrate concentrations and different rate constants are generated. A comparison of these curves with the curves obtained from the rapid equilibrium initial rate equation, with different substrate concentration values, serves to analyze how the strict steady-state rate equation values are closer to those of rapid equilibrium rate equations when rapid equilibrium conditions are fulfilled.

scholarly journals The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step

1989 ◽  
Vol 259 (3) ◽  
pp. 709-713 ◽  
Author(s):  
E Rigney ◽  
T J Mantle ◽  
F M Dickinson
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Protein Fluorescence ◽  
Biliverdin Reductase ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a ‘burst’ was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the ‘burst’. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state ‘burst’ reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.

scholarly journals Transient kinetics of nicotinamide–adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria

1978 ◽  
Vol 171 (3) ◽  
pp. 743-750 ◽  
Author(s):  
K Dalziel ◽  
N McFerran ◽  
B Matthews ◽  
C H Reynolds
Keyword(s):  
Steady State ◽  
Isocitrate Dehydrogenase ◽  
Turnover Rate ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Lag Phase ◽  
Transient Kinetics ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.

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