scholarly journals Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma

1994 ◽  
Vol 301 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S Lyle ◽  
D H Geller ◽  
K Ng ◽  
J Westley ◽  
N B Schwartz
Keyword(s):  
Steady State ◽  
Initial Velocity ◽  
Kinase Activity ◽  
Forward Direction ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Reverse Direction ◽  
Aps Kinase ◽  
Ordinate Axis ◽  
Atp Sulphurylase

ATP-sulphurylase catalyses the production of adenosine 5′-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3′-phosphate 5′-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.

scholarly journals Kinetic mechanism of adenosine 5′-phosphosulphate kinase from rat chondrosarcoma

1994 ◽  
Vol 301 (2) ◽  
pp. 355-359 ◽  
Author(s):  
S Lyle ◽  
D H Geller ◽  
K Ng ◽  
J Stanczak ◽  
J Westley ◽  
...  
Keyword(s):  
Steady State ◽  
Initial Velocity ◽  
Mixed Type ◽  
Competitive Inhibition ◽  
Kinetic Mechanism ◽  
Sequential Action ◽  
Aps Kinase ◽  
Formal Mechanism ◽  
Inhibition Studies ◽  
Atp Sulphurylase

Biosynthesis of the activated sulphate donor adenosine 3′-phosphate 5′-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5′-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3′-phosphate 5′-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5′-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.

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 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 Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase

1978 ◽  
Vol 175 (3) ◽  
pp. 987-998 ◽  
Author(s):  
A Lodola ◽  
J D Shore ◽  
D M Parker ◽  
J Holbrook
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Forward Direction ◽  
Divergent Evolution ◽  
Reverse Direction ◽  
Enzyme Form ◽  
Active Substrate ◽  
Rate Limiting ◽  
Transient Measurements

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows ‘all-of-the-sites’ behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.

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.

Steady-State Kinetic Mechanism of Recombinant Avocado ACC Oxidase:  Initial Velocity and Inhibitor Studies†

Biochemistry ◽  
2000 ◽  
Vol 39 (35) ◽  
pp. 10730-10738 ◽  
Author(s):  
Norbert M. W. Brunhuber ◽  
Jessica L. Mort ◽  
Rolf E. Christoffersen ◽  
Norbert O. Reich
Keyword(s):  
Steady State ◽  
Initial Velocity ◽  
Acc Oxidase ◽  
Kinetic Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 139 (1) ◽  
pp. 27-35 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Exchange Reaction ◽  
Initial Rate ◽  
Leaf Tissue ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Sequential Mechanism ◽  
Spinach Leaf ◽  
Atp Sulphurylase

1. Sulphate-dependent PPi–ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP2− and MgP2O72−; ATP sulphurylase activity was not correlated with the concentration of free Mg2+. 2. Sulphate-dependent PPi–ATP exchange was independent of PPi concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PPi–ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [32P]ATP from [32P]PPi and adenosine 5′-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PPi and adenosine 5′-sulphatophosphate. 5. The synthesis of ATP from PPi and adenosine 5′-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PPi and adenosine 5′-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5′-sulphatophosphate and non-competitive with respect to PPi. It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP2− was the first product to react with the enzyme and MgP2O72− was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5′-sulphatophosphate could not be demonstrated.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

scholarly journals Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

1988 ◽  
Vol 250 (1) ◽  
pp. 25-31 ◽  
Author(s):  
C MacKintosh ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Intact Cells ◽  
Competing Anions ◽  
Effects Of Ph ◽  
Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

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