scholarly journals Kinetic evidence for the formation of a Michaelis–Menten-like complex between horseradish peroxidase compound II and di-(N-acetyl-l-tyrosine)

1999 ◽  
Vol 340 (1) ◽  
pp. 329-336 ◽  
Author(s):  
Weichi WANG ◽  
Stéphanie NOËL ◽  
Michel DESMADRIL ◽  
Jacques GUÉGUEN ◽  
Thierry MICHON
Keyword(s):  
Steady State ◽  
Horseradish Peroxidase ◽  
Adsorption Complex ◽  
Flow Measurements ◽  
K Value ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Steady State Kinetic ◽  
Compound Ii ◽  
Rate Limiting

The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-ʟ-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific K value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2 → (NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (k/K≈ kII → E). The k values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The K value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific K value for the second step of reduction (CII → E) and to its thermodynamic dissociation constant (Kd = 0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.

scholarly journals Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases

2021 ◽  
Vol 22 (5) ◽  
pp. 2629
Author(s):  
Dolores Linde ◽  
Iván Ayuso-Fernández ◽  
Marcos Laloux ◽  
José E. Aguiar-Cervera ◽  
Antonio L. de Lacey ◽  
...  
Keyword(s):  
Veratryl Alcohol ◽  
Flow Measurements ◽  
Reduction Potentials ◽  
Rate Limiting Step ◽  
Lower Extent ◽  
Lignin Model ◽  
Bacteria And Fungi ◽  
Compound Ii ◽  
Rate Limiting ◽  
Lignin Macromolecule

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-β- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s−1·mM−1 for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1–3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28–1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis

2016 ◽  
Vol 473 (5) ◽  
pp. 651-660 ◽  
Author(s):  
Renata A.G. Reis ◽  
Patricia Ferreira ◽  
Milagros Medina ◽  
M. Cristina Nonato
Keyword(s):  
Steady State ◽  
Leishmania Major ◽  
De Novo ◽  
Enzyme Reaction ◽  
Mechanistic Study ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Rate Limiting ◽  
State Kinetic

Leishmania major dihydro-orotate dehydrogenase (DHODHLm) oxidizes dihydro-orotate to orotate (ORO) in the de novo pyrimidine biosynthetic pathway. The enzyme reaction mechanism was elucidated by steady- and pre-steady-state kinetics. ORO release was found to be the rate-limiting step in the overall catalysis.

scholarly journals Phenothiazines are slowly oxidizable substrates of horseradish peroxidase

2011 ◽  
Vol 57 (5) ◽  
pp. 544-553 ◽  
Author(s):  
T.V. Rogozhina ◽  
V.V. Rogozhin
Keyword(s):  
Steady State ◽  
Horseradish Peroxidase ◽  
Reaction Medium ◽  
Substrate Oxidation ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Full Conversion ◽  
Peroxidase Oxidation

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. kcat and Km values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH>5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.

scholarly journals Kinetics and mechanism of action of muscle pyruvate kinase

1978 ◽  
Vol 169 (1) ◽  
pp. 39-54 ◽  
Author(s):  
Leighton G. Dann ◽  
Hubert G. Britton
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Pyruvate Kinase ◽  
Alternative Pathway ◽  
Slow Step ◽  
Velocity Data ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Muscle Pyruvate Kinase ◽  
Rate Limiting

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg2+. 2. Methods of preparing [32P]phosphoenolpyruvate from [32P]Pi in high yield and determining [32P]-phosphoenolpyruvate and [8-14C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [32P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-14C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [32P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg2+, with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-14C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-14C]-pyruvate or [γ-32P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

Antithrombin - Mechanism Of Action And Binding Of Heparin

1981 ◽  
Author(s):  
I Björk ◽  
U Lindahl
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inactivation ◽  
Substrate Complex ◽  
High Affinity ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Rate Limiting

Antithrombin inhibits a variety of serine proteases by forming equimolar, inactive complexes with the enzymes. The anti thrombin-thrombin complex, extensively studied as a model for complexes with other coagulation proteases, dissociates with a half-life of several days to free enzyme and a proteolytically modified inhibitor. It thus behaves like a kinetically stable enzyme-substrate complex. Several observations indicate that deacylation is the rate-limiting step. The active site of antithrombin, i.e. the bond slowly cleaved by the target enzyme, is the Arg-385/Ser-386 bond in the carboxy-terminal region of the protein. The formation of most anti thrombin-protease complexes is greatly accelerated by certain forms of heparin. These active molecules comprise about 1/3 of normal heparin preparations and bind with high affinity (K∼108 M-1) to the inhibitor, regardless of the size of the polysaccharide. The stoichiometry of binding is 1:1 for most heparin molecules, although some high-molecular-weight chains have two antithrombin binding sites. Evidence from spectroscopic and kinetic analyses suggests that the binding of high-affinity heparin induces a conformational change in antithrombin that probably is involved in the mechanism of the increased rate of enzyme inactivation. Oligosaccharides with high-affinity for anti thrombin have been isolated by affinity chromatography following partial deaminative cleavage of heparin with nitrous acid. The smallest such oligosaccharide obtained is an octasaccharide, in which a pentasaccharide sequence appears to comprize the actual antithrombin-binding site. This active sequence contains a unique, 3-O-sulfated glucosamine residue that does not appear to occur in other portions of the heparin molecule. In addition, two N-sulfate groups and probably at least one O-sulfate group within the pentasaccharide sequence are essential for high-affinity binding of heparin to antithrombin.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

scholarly journals Characterization of a Baculovirus-Encoded RNA 5′-Triphosphatase

1998 ◽  
Vol 72 (9) ◽  
pp. 7057-7063 ◽  
Author(s):  
Christian H. Gross ◽  
Stewart Shuman
Keyword(s):  
Steady State ◽  
Product Formation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Mrna Capping ◽  
Steady State Rate ◽  
Rna Triphosphatase ◽  
State Rate ◽  
Nuclear Polyhedrosis ◽  
Rate Limiting

ABSTRACT Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5′-triphosphatase that hydrolyzes the γ-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 μM γ-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min−1 and converts ATP to ADP with V max of 8 min−1and Km of 25 μM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5′ ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.

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