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

1970 ◽  
Vol 48 (5) ◽  
pp. 594-603 ◽  
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.

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 studies with the low-Km aldehyde reductase from ox brain

1985 ◽  
Vol 227 (2) ◽  
pp. 621-627 ◽  
Author(s):  
C M Ryle ◽  
K F Tipton
Keyword(s):  
Initial Velocity ◽  
Initial Rate ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Binary Complex ◽  
Aldehyde Reductase ◽  
Apparent Dissociation Constant ◽  
Displacement Mechanism ◽  
Sequential Mechanism ◽  
Inhibition Studies

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

Kinetic Studies of Phosphoribosyl-formylglycineamidine Synthetase

1972 ◽  
Vol 50 (5) ◽  
pp. 490-500 ◽  
Author(s):  
Samuel Y. Chu ◽  
J. Frank Henderson
Keyword(s):  
Ammonium Chloride ◽  
Initial Velocity ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Ping Pong ◽  
Inhibition Constants ◽  
Inhibition Studies

Initial velocity and product inhibition studies of phosphoribosyl-formylglycineamidine synthetase indicate that the reaction involves a fully ping pong mechanism in which glutamine binds to the free enzyme and glutamate is released before the addition of ATP. ADP is released, and phosphoribosyl-formylglycineamide then binds; the liberation of Pi is rapid, and phosphoribosyl-formylglycineamidine is the last product released from the enzyme. The Km values for glutamine, ATP, and phosphoribosyl-formylglycineamide are 1.1 × 10−4 M, 1.5 × 10−3 M, and 1.1 × 10−4 M, respectively. The Km value for ammonium chloride is 7.5 × 10−3 M, and the ratio of Vmax values with ammonium chloride and glutamine is 1/40. The inhibition constants for FGAM and Pi were calculated to be 1.3 × 10−4 M and 6.45 × 10−3 M, respectively.

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

2007 ◽  
Vol 404 (3) ◽  
pp. 439-448 ◽  
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.

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.

Kinetic mechanism of a recombinant Arabidopsis glyoxylate reductase: studies of initial velocity, dead-end inhibition and product inhibition

2007 ◽  
Vol 85 (9) ◽  
pp. 896-902 ◽  
Author(s):  
Gordon J. Hoover ◽  
Gerald A. Prentice ◽  
A. Rod Merrill ◽  
Barry J. Shelp
Keyword(s):  
Initial Velocity ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Succinic Semialdehyde ◽  
In Planta ◽  
Dead End ◽  
Arabidopsis Protein ◽  
Enzyme Functions ◽  
Glyoxylate Reductase

Kinetic analysis of substrate specificity revealed that a recombinant Arabidopsis protein catalyzes the conversion of glyoxylate to glycolate (Km,glyoxylate = 4.5 μmol·L–1) and succinic semialdehyde (SSA) to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L–1) via an essentially irreversible, NADPH-based mechanism. In this report, the enzyme was further characterized via initial-velocity, dead-end inhibition and product inhibition studies. The kinetic mechanism was ordered Bi Bi, involving the complexation of NADPH to the enzyme before glyoxylate or SSA, and the release of NADP+ before glycolate or γ-hydroxybutyrate, respectively. It can be concluded that the enzyme functions as a NADPH-dependent glyoxylate reductase (EC 1.1.1.79) or possibly an aldehyde reductase (EC 1.1.1.2), and the kinetic mechanism involved is consistent with that found in members of both the aldo-keto reductase and 3-hydroxyisobutyrate dehydrogenase-related superfamilies of enzymes. Since NADP+ was an effective competitive inhibitor with respect to NADPH (Ki = 1–3 µmol·L–1), it is proposed that the ratio of NADPH/NADP+ regulates enzymatic activity in planta.

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.

Sign in / Sign up

Export Citation Format

Share Document