scholarly journals The kinetic mechanism of the major form of ox kidney aldehyde reductase with d-glucuronic acid

1982 ◽  
Vol 205 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ann K. Daly ◽  
Timothy J. Mantle
Keyword(s):  
Glucuronic Acid ◽  
Alternative Pathway ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Aldehyde Reductase ◽  
Major Form ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Inhibition Studies ◽  
Kinetics Of

The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

scholarly journals Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

scholarly journals Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase

1987 ◽  
Vol 242 (1) ◽  
pp. 143-150 ◽  
Author(s):  
K S De Jongh ◽  
P J Schofield ◽  
M R Edwards
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Free Enzyme ◽  
Aldehyde Reductase ◽  
Binding Studies ◽  
Sheep Liver ◽  
Low Concentrations ◽  
High Concentrations ◽  
Inhibition Studies

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.

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 Comparison of the active sites of the purified carnitine acyltransferases from peroxisomes and mitochondria by using a reaction-intermediate analogue

1993 ◽  
Vol 294 (3) ◽  
pp. 645-651 ◽  
Author(s):  
N Nic a′ Bháird ◽  
G Kumaravel ◽  
R D Gandour ◽  
M J Krueger ◽  
R R Ramsay
Keyword(s):  
Active Sites ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Binding Domains ◽  
Cell Compartments ◽  
Mitochondrial Inner Membrane ◽  
Kinetic Mechanisms ◽  
Carnitine Acyltransferases ◽  
Inhibition Studies

The carnitine acyltransferases contribute to the modulation of the acyl-CoA/CoA ratio in various cell compartments with consequent effects on many aspects of fatty acid metabolism. The properties of the enzymes are different in each location. The kinetic mechanisms and kinetic parameters for the carnitine acyltransferases purified from peroxisomes (COT) and from the mitochondrial inner membrane (CPT-II) were determined. Product-inhibition studies established that COT follows a rapid-equilibrium random-order mechanism, but CPT-II follows a strictly ordered mechanism in which acyl-CoA or CoA must bind before the carnitine substrate. Hemipalmitoylcarnitinium [(+)-HPC], a prototype tetrahedral intermediate analogue of the acyltransferase reaction, inhibits CPT-II 100-fold better than COT. (+)-HPC behaves as an analogue of palmitoyl-L-carnitine with COT. In contrast, with CPT-II(+)-HPC binds more tightly to the enzyme than do substrates or products, suggesting that it is a good model for the transition state and, unlike palmitoyl-L-carnitine, (+)-HPC can bind to the free enzyme. The data support the concept of three binding domains for the acyltransferases, a CoA site, an acyl site and a carnitine site. The CoA site is similar in COT and CPT-II, but there are distinct differences between the carnitine-binding site which may dictate the kinetic mechanism.

scholarly journals Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate

1986 ◽  
Vol 234 (2) ◽  
pp. 317-323 ◽  
Author(s):  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Isocitrate Dehydrogenase ◽  
Initial Rate ◽  
Potent Inhibitor ◽  
Competitive Inhibition ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Coenzyme Binding ◽  
Inhibition Studies

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.

scholarly journals Kinetic mechanism of pulmonary carbonyl reductase

1988 ◽  
Vol 252 (1) ◽  
pp. 17-22 ◽  
Author(s):  
K Matsuura ◽  
T Nakayama ◽  
M Nakagawa ◽  
A Hara ◽  
H Sawada
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Carbonyl Reductase ◽  
Ternary Complexes ◽  
Enzyme Mechanism ◽  
The Other ◽  
Binding Studies ◽  
Forward Reaction ◽  
Cibacron Blue ◽  
Inhibition Studies

The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes.

scholarly journals d-3-Hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose

1973 ◽  
Vol 133 (1) ◽  
pp. 133-157 ◽  
Author(s):  
M. J. Preuveneers ◽  
D. Peacock ◽  
E. M. Crook ◽  
J. B. Clark ◽  
K. Brocklehurst
Keyword(s):  
Rate Constants ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Covalent Attachment ◽  
Soluble Enzyme ◽  
Steady State Kinetics ◽  
Dead End ◽  
Rhodopseudomonas Spheroides

1. The reversible NAD+-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0°C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate–NAD+ oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2μmol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme–nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme–nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.

scholarly journals Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea

1989 ◽  
Vol 67 (11-12) ◽  
pp. 763-769 ◽  
Author(s):  
Emidio De Carolis ◽  
Ragai K. Ibrahim
Keyword(s):  
Competitive Inhibition ◽  
Divalent Cations ◽  
Product Inhibition ◽  
Affinity Column ◽  
Ph Optimum ◽  
Substrate Interaction ◽  
Interaction Kinetics ◽  
Molecular Masses ◽  
Inhibition Studies ◽  
Kinetics Of

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.

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