Kinetic mechanism of pulmonary carbonyl reductase

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.

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Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase

Biochemical Journal ◽

10.1042/bj2420143 ◽

1987 ◽

Vol 242 (1) ◽

pp. 143-150 ◽

Cited By ~ 19

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.

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

Biochemical Journal ◽

10.1042/bj2050381 ◽

1982 ◽

Vol 205 (2) ◽

pp. 381-388 ◽

Cited By ~ 12

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.

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Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

Biochemical Journal ◽

10.1042/bj2610935 ◽

1989 ◽

Vol 261 (3) ◽

pp. 935-943 ◽

Cited By ~ 11

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.

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

Biochemical Journal ◽

10.1042/bj2940645 ◽

1993 ◽

Vol 294 (3) ◽

pp. 645-651 ◽

Cited By ~ 21

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.

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

Biochemical Journal ◽

10.1042/bj2970327 ◽

1994 ◽

Vol 297 (2) ◽

pp. 327-333 ◽

Cited By ~ 18

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.

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Phosphopantetheine Adenylyltransferase from Escherichia coli: Investigation of the Kinetic Mechanism and Role in Regulation of Coenzyme A Biosynthesis

Journal of Bacteriology ◽

10.1128/jb.00732-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8196-8205 ◽

Cited By ~ 26

Author(s):

J. Richard Miller ◽

Jeffrey Ohren ◽

Ronald W. Sarver ◽

W. Thomas Mueller ◽

Piet de Dreu ◽

...

Keyword(s):

Escherichia Coli ◽

Ligand Binding ◽

Coenzyme A ◽

Forward Direction ◽

Kinetic Mechanism ◽

Product Inhibition ◽

Titration Calorimetry ◽

Antibacterial Drug ◽

Binding Modes ◽

Binding Studies

ABSTRACT Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.

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

Biochemical Journal ◽

10.1042/bj2340317 ◽

1986 ◽

Vol 234 (2) ◽

pp. 317-323 ◽

Cited By ~ 15

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.

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Kinetic analysis of duck ε-crystallin, a lens structural protein with lactate dehydrogenase activity

Biochemical Journal ◽

10.1042/bj2670051 ◽

1990 ◽

Vol 267 (1) ◽

pp. 51-58 ◽

Cited By ~ 17

Author(s):

S H Chiou ◽

H J Lee ◽

G G Chang

Keyword(s):

Lactate Dehydrogenase ◽

Kinetic Analysis ◽

Substrate Inhibition ◽

Product Inhibition ◽

Enzyme Mechanism ◽

Kinetic Properties ◽

Structural Protein ◽

Lactate Dehydrogenase Activity ◽

Chicken Heart ◽

Inhibition Studies

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.

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Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj20061815 ◽

2007 ◽

Vol 404 (3) ◽

pp. 439-448 ◽

Cited By ~ 39

Author(s):

Katherine M. Brendza ◽

William Haakenson ◽

Rebecca E. Cahoon ◽

Leslie M. Hicks ◽

Lavanya H. Palavalli ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Growth And Development ◽

Initial Velocity ◽

Kinetic Mechanism ◽

Product Inhibition ◽

Parasitic Nematodes ◽

Rnai Phenotype ◽

C Elegans ◽

Inhibition Studies ◽

Single Enzyme

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.

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Kinetic studies of sulfite; cytochrome c oxidoreductase, thiosulfate-oxidizing enzyme, and adenostne-5′-phosphosulfate reductase from Thiobacillus thioparus

Canadian Journal of Biochemistry ◽

10.1139/o70-097 ◽

1970 ◽

Vol 48 (5) ◽

pp. 594-603 ◽

Cited By ~ 5

Author(s):

Ronald M. Lyric ◽

Isamu Suzuki

Keyword(s):

Cytochrome C ◽

Initial Velocity ◽

Enzyme Reaction ◽

Kinetic Studies ◽

Kinetic Mechanism ◽

Product Inhibition ◽

Thiobacillus Thioparus ◽

Inhibition Studies

Kinetic studies were carried out on three enzymes purified from Thiobacillus thioparus: sulfite: cytochrome c oxidoreductase, thiosulfate-oxidizing enzyme, and adenosine-5′-phosphosulfate reductase. From the initial velocity and product inhibition studies a tentative kinetic mechanism was proposed for each enzyme reaction.

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