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 Kinetic mechanism of the dimeric ATP sulfurylase from plants

2013 ◽  
Vol 33 (4) ◽  
Author(s):  
Geoffrey E. Ravilious ◽  
Jonathan Herrmann ◽  
Soon Goo Lee ◽  
Corey S. Westfall ◽  
Joseph M. Jez
Keyword(s):  
Initial Velocity ◽  
Competitive Inhibition ◽  
Tight Binding ◽  
Atp Synthesis ◽  
Kinetic Mechanism ◽  
Titration Calorimetry ◽  
Atp Sulfurylase ◽  
Dead End ◽  
Sequential Mechanism ◽  
Enzyme Functions

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.

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 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 of rat liver hexokinase D (glucokinase) in non-co-operative conditions show an ordered mechanism with MgADP as the last product to be released

2003 ◽  
Vol 371 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Octavio MONASTERIO ◽  
María Luz CÁRDENAS
Keyword(s):  
Rat Liver ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Nitrobenzoic Acid ◽  
Binary Complexes ◽  
Dead End ◽  
Straight Line ◽  
Inactivation Constant ◽  
Reciprocal Value

The kinetic mechanism of rat liver hexokinase D ('glucokinase') was studied under non-co-operative conditions with 2-deoxyglucose as substrate, chosen to avoid uncertainties derived from the co-operativity observed with the physiological substrate, glucose. The enzyme shows hyperbolic kinetics with respect to both 2-deoxyglucose and MgATP2-, and the reaction follows a ternary-complex mechanism with Km = 19.2±2.3mM for 2-deoxyglucose and 0.56±0.05mM for MgATP2-. Product inhibition by MgADP- was mixed with respect to MgATP2- and was largely competitive with respect to 2-deoxyglucose, suggesting an ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. Dead-end inhibition by N-acetylglucosamine, AMP and the inert complex CrATP [the complex of ATP with chromium in the 3+ oxidation state, i.e. Cr(III)—ATP], studied with respect to both substrates, also supports an ordered mechanism with 2-deoxyglucose as first substrate. AMP appears to bind both to the free enzyme and to the E·dGlc complex. Experiments involving protection against inactivation by 5,5′-dithiobis-(2-nitrobenzoic acid) support the existence of the E·MgADP- and E·AMP complexes suggested by the kinetic studies. MgADP-, AMP, 2-deoxyglucose, glucose and mannose were strong protectors, supporting the existence of binary complexes with the enzyme. Glucose 6-phosphate failed to protect, even at concentrations as high as 100mM, and MgATP2- protected only slightly (12%). The inactivation results support the postulated ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. In addition, the straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugar-binding site in hexokinase D.

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 Identification of the kinetic mechanism of succinyl-CoA synthetase

2013 ◽  
Vol 33 (1) ◽  
Author(s):  
Xin Li ◽  
Fan Wu ◽  
Daniel A. Beard
Keyword(s):  
Experimental Data ◽  
Catalytic Mechanism ◽  
Model Simulation ◽  
Tricarboxylic Acid Cycle ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Catalysed Reaction ◽  
Dead End ◽  
Haem Biosynthesis ◽  
Parameter Values

The kinetic mechanism of SCS [succinyl-CoA (coenzyme A) synthetase], which participates in the TCA (tricarboxylic acid) cycle, ketone body metabolism and haem biosynthesis, has not been fully characterized. Namely, a representative catalytic mechanism and associated kinetic parameters that can explain data on the enzyme-catalysed reaction kinetics have not been established. To determine an accurate model, a set of putative mechanisms of SCS, proposed by previous researchers, were tested against experimental data (from previous publication) on SCS derived from porcine myocardium. Based on comparisons between model simulation and the experimental data, an ordered ter–ter mechanism with dead-end product inhibition of succinate against succinyl-CoA is determined to be the best candidate mechanism. A thermodynamically constrained set of parameter values is identified for this candidate mechanism.

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.

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 The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase

1965 ◽  
Vol 97 (1) ◽  
pp. 37-52 ◽  
Author(s):  
JF Morrison ◽  
E James
Keyword(s):  
Initial Velocity ◽  
Dissociation Constants ◽  
Product Inhibition ◽  
Ternary Complexes ◽  
The Other ◽  
Velocity Pattern ◽  
Dead End ◽  
Rapid Equilibrium ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.

scholarly journals Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase

1992 ◽  
Vol 286 (1) ◽  
pp. 243-251 ◽  
Author(s):  
N L Deans ◽  
R D Allison ◽  
D L Purich
Keyword(s):  
Steady State ◽  
Thermal Inactivation ◽  
Enzyme Reaction ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Maximal Velocity ◽  
Steady State Kinetic ◽  
Tubulin Tyrosine Ligase ◽  
State Kinetic

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.

Sign in / Sign up

Export Citation Format

Share Document