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.

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 Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes

1975 ◽  
Vol 151 (3) ◽  
pp. 715-727 ◽  
Author(s):  
M J Boland ◽  
H Gutfreund
Keyword(s):  
Lactate Dehydrogenase ◽  
Dissociation Constant ◽  
Turnover Rate ◽  
Nadh Oxidation ◽  
Lactate Oxidation ◽  
First Order ◽  
Single Turnover ◽  
Ph Values ◽  
Km Value ◽  
Rate Limiting

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.

scholarly journals Kinetic Studies of Rabbit Muscle Lactate Dehydrogenase

1962 ◽  
Vol 237 (5) ◽  
pp. 1668-1675
Author(s):  
Virginia Zewe ◽  
Herbert J. Fromm
Keyword(s):  
Lactate Dehydrogenase ◽  
Kinetic Studies ◽  
Rabbit Muscle ◽  
Muscle Lactate

scholarly journals Kinetic studies of oxidized nicotinamide–adenine dinucleotide-facilitated reactions of d-glyceraldehyde 3-phosphate dehydrogenase

1974 ◽  
Vol 143 (2) ◽  
pp. 353-363 ◽  
Author(s):  
Patricia J. Harrigan ◽  
David R. Trentham
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Temperature Jump ◽  
Reaction Pathway ◽  
Kinetic Studies ◽  
Rate Equations ◽  
First Order ◽  
Diffusion Controlled ◽  
Relaxation Temperature ◽  
Pig Muscle ◽  
Kinetics Of

The kinetics of the acylation of d-glyceraldehyde 3-phosphate dehydrogenase from pig muscle by 1,3-diphosphoglycerate in the presence of NAD+ has been analysed by using the relaxation temperature-jump method. At pH7.2 and 8°C the rate of acylation of the NAD+-bound (or holo-) enzyme was 3.3×105m−1·s−1 and the rate of phosphorolysis, the reverse reaction, was 7.5×103m−1·s−1. After a temperature-jump perturbation the equilibrium of NAD+ binding to the acyl-enzyme was re-established more rapidly than that of the acylation. The rate of phosphorolysis of the apoacylenzyme from sturgeon muscle and of aldehyde release from the d-glyceraldehyde 3-phosphate–apoenzyme complex were ≤40m−1·s−1 and ≤12s−1 respectively at pH8.0 and 22°C, which means that both processes are too slow to contribute significantly to the reaction pathway of the reversible NAD+-linked oxidative phosphorylation of d-glyceraldehyde 3-phosphate. Phosphorolysis of both acyl-apoenzyme and acyl-holoenzyme was first-order in Pi up to 100mm-Pi and more. PO43− could be the reactive species of the phosphorolysis of the acyl-holoenzyme, in which case phosphorolysis is a diffusion-controlled reaction, although other kinetically indistinguishable rate equations for the reaction are possible.

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.

scholarly journals Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase

1978 ◽  
Vol 175 (3) ◽  
pp. 987-998 ◽  
Author(s):  
A Lodola ◽  
J D Shore ◽  
D M Parker ◽  
J Holbrook
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Forward Direction ◽  
Divergent Evolution ◽  
Reverse Direction ◽  
Enzyme Form ◽  
Active Substrate ◽  
Rate Limiting ◽  
Transient Measurements

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows ‘all-of-the-sites’ behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.

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