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 Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 533-538 ◽  
Author(s):  
A K H MacGibbon ◽  
S J Haylock ◽  
P D Buckley ◽  
L F Blackwell
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
Michaelis Constants ◽  
State Rate ◽  
Liver Aldehyde Dehydrogenase

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

scholarly journals Further studies of the action of disulfiram and 2,2′-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 203 (3) ◽  
pp. 743-754 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Chloral Hydrate ◽  
Enzyme Molecule ◽  
Catalysed Reaction ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
State Rate ◽  
Usual Site ◽  
Esterase Activities

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2′-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2′-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.

scholarly journals Characterization of a Baculovirus-Encoded RNA 5′-Triphosphatase

1998 ◽  
Vol 72 (9) ◽  
pp. 7057-7063 ◽  
Author(s):  
Christian H. Gross ◽  
Stewart Shuman
Keyword(s):  
Steady State ◽  
Product Formation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Mrna Capping ◽  
Steady State Rate ◽  
Rna Triphosphatase ◽  
State Rate ◽  
Nuclear Polyhedrosis ◽  
Rate Limiting

ABSTRACT Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5′-triphosphatase that hydrolyzes the γ-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 μM γ-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min−1 and converts ATP to ADP with V max of 8 min−1and Km of 25 μM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5′ ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.

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.

scholarly journals Studies on the unusual behaviour of bovine liver UDP-glucose dehydrogenase in assays at acid and neutral pH and on the presence of tightly bound nucleotide material in purified preparations of this enzyme

1988 ◽  
Vol 255 (3) ◽  
pp. 775-780 ◽  
Author(s):  
F M Dickinson
Keyword(s):  
Steady State ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Enzyme Release ◽  
Unusual Behaviour ◽  
Ph Values ◽  
Steady State Rate ◽  
State Rate ◽  
Slow Dissociation ◽  
Enzyme Species

Assays of UDP-glucose dehydrogenase at pH 6.0 show long (10-15 min) lag periods before the steady-state rate is established, but at pH 9.0 no lag is observed. At intermediate pH values the lag is progressively shorter as the pH becomes more alkaline. The behaviour of the enzyme in assays at neutral and acid pH depends on the pH and concentration of the enzyme used to initiate the assay. The steady-state rate at pH 6.0 is strongly concentration-dependent. It is suggested that these phenomena arise because of the slow dissociation of an inactive enzyme species to an active one. Purified preparations of the enzyme release approx. 1 mol of a UDP-sugar/mol of enzyme subunit on denaturation. The identity of the UDP-sugar is unknown.

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.

scholarly journals A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

1996 ◽  
Vol 316 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Trevor M. KITSON ◽  
Kathryn E. KITSON
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Visible Region ◽  
Stopped Flow ◽  
Site Model ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Reporter Group ◽  
Hydrolysis Of

1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in rate-determining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient ‘reporter group’ produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7–7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual ‘negative burst’ in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

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.

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.

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