scholarly journals The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

1976 ◽  
Vol 159 (3) ◽  
pp. 513-527 ◽  
Author(s):  
J M Armstrong ◽  
D R Trentham
Keyword(s):  
Rate Constant ◽  
Active Sites ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Rate Equations ◽  
Buffer System ◽  
Exponential Process ◽  
Order Rate ◽  
Rate Profile

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 × 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 × 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

Studies on Horseradish Peroxidase. XI. On the Nature of Compounds I and II as Determined from the Kinetics of the Oxidation of Ferrocyanide

1973 ◽  
Vol 51 (4) ◽  
pp. 582-587 ◽  
Author(s):  
M. L. Cotton ◽  
H. B. Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Rate Constant ◽  
Active Sites ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Compound I ◽  
Order Rate ◽  
Compound Ii ◽  
Kinetics Of

In order to investigate the nature of compounds I and II of horseradish peroxidase, the kinetics were studied of ferrocyanide oxidation catalyzed by these compounds which were prepared from three different oxidizing agents. The pH dependence of the apparent second-order rate constant for ferrocyanide oxidation by compound I, prepared from ethyl hydroperoxide and m-chloroperbenzoic acid, was interpreted in terms of an ionization on the enzyme with a pKa = 5.3, identical to that reported previously for hydrogen peroxide. The second-order rate constant for the compound II-ferrocyanide reaction also showed the same pH dependence for the three oxidizing substrates. However, with more accurate results, the compound II-ferrocyanide reaction was reinterpreted in terms of a single ionization with pKa = 8.5. The same dependence of ferrocyanide oxidation on pH suggests structurally identical active sites for compounds I and II prepared from the three different oxidizing substrates.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

scholarly journals Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain

1992 ◽  
Vol 287 (3) ◽  
pp. 881-889 ◽  
Author(s):  
M Patel ◽  
I S Kayani ◽  
W Templeton ◽  
G W Mellor ◽  
E W Thomas ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Molecular Recognition ◽  
Rate Constant ◽  
Kinetic Data ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Catalytic Site ◽  
Order Rate ◽  
S2 Subsite

1. 2-(N′-Acetyl-D-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound IV) (m.p. 59 degrees C; [alpha]D20 -6.6 degrees (c 1.2 in methanol)) was synthesized. 2. The results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group (Cys-25) of papain (EC 3.4.22.2) together with analogous kinetic data for the reactions of related time-dependent inhibitors, notably the L-enantiomer of compound (IV) (compound III) and the L- and D-enantiomers of 2-(N′-acetylphenylalanylamino)ethyl 2′-pyridyl disulphide (compounds I and II respectively), were used to assess the contributions of the (P1)-NH ... O = C < (Asp-158) and (P2) > C = O ... H-N-(Gly-66) hydrogen bonds and enantiomeric P2-S2 hydrophobic contacts in two manifestations of dynamic molecular recognition in papain-ligand association: (a) signalling to the catalytic-site region to provide for a (His-159)-IM(+)-H-assisted transition state and (b) the dependence of P2-S2 stereoselectivity on hydrogen-bonding interactions outside the S2 subsite. The analysis involved determination of the reactivities of individual ionization states of the reactions (pH-independent rate constants, k) and associated macroscopic pKa values and difference kinetic specificity energies (delta delta GKS = -RT1n(k1/k2), where k1 is the pH-independent second-order rate constant for reaction with one inhibitor and k2 is the analogous rate constant in the same ionization state for reaction with another inhibitor so that, when the structural change provides that k2 > k1, delta delta GKS is positive. 3. The kinetic data further illuminate the nature of the interdependence of binding interactions in papain first noted by Kowlessur, Topham, Thomas, O'Driscoll, Templeton & Brocklehurst [(1989) Biochem. J. 258, 755-764] in the S2 subsite, S1-S2 intersubsite and catalytic-site regions. Of particular note is the apparent dependence of the binding of the N-Ac-D-Phe moiety on the binding of the leaving group to (His-159)-Im+H and the fact that the resulting rate enhancement is more effective when (P1)-N-H is absent than when it is present. This result revealed by kinetic analysis goes beyond the conclusion suggested by model building that it is possible to make all of the binding contacts in complexes involving the D-enantiomers [(II) and (IV)] as in those involving the L-enantiomers [(I) and (III)].(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values

1977 ◽  
Vol 167 (3) ◽  
pp. 859-862 ◽  
Author(s):  
K Brocklehurst ◽  
H B F Dixon
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Quasi Equilibrium ◽  
Pka Values ◽  
First Order ◽  
Order Rate ◽  
Parallel Reactions

1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix.

DFP (Dilsopropylfluorophosphate) Studies on Molecular Mechanisms of Surface-Dependent Activation of Prekallikrein(PK) and Factor XII(Hageman Factor, HF)

1979 ◽  
Author(s):  
J.H. Griffin ◽  
G. Beretta
Keyword(s):  
Enzymatic Activity ◽  
Rate Constant ◽  
Active Sites ◽  
Molecular Mechanisms ◽  
Order Rate Constant ◽  
Factor Xii ◽  
Single Chain ◽  
Hageman Factor ◽  
Order Rate ◽  
Coagulant Activity

Previous 3H-DFP studies showed that HF binding to kaolin does not result in formation of new active sites. New studies show that single chain zymogen HF, in solution or surface bound, reacts slowly with DFP causing loss of coagulant activity and uptake of 1 mol DIP/mol HF with a 2nd order rate constant, k2=0.4 M-1 min-1. High MW kininogen (HMWK) did not affect the rate constant for this reaction. PK reacts with DFP causing loss of coagulant activity with k2=0.6 M-1min-1. These values are ~103lower than for the activated enzymes (160 and 500 M-1 min-1 for HFa and kallikrein respectively) and are similar to values for trypsinogen that exhibits weak enzymatic activity. HF, PK, HMWK, and kaolin were separately preincubated with 40mM DFP for 5 min to inhibit traces of active enzymes. Then, mixing of these reagents in the presence of 40mM DFP caused a burst of cleavage of 131I-HF and 125I-PK, the extent of which depended on the amount of HMWK. To avoid reciprocal proteolysis of HF and PK, “killed” zymogens (DIP-HF or DIP-PK formed by 48 hr incubation with 40mM DFP) were used in place of HF or PK. Controls showed that PK and DIP-PK were similarly cleaved by purified HFa. If DIP-131I-HF or DIP-125I-PK was substituted for HF or PK in mixtures of HF, PK, HMWK, and kaolin, no detectable cleavage occurred. These data allow that inherent activity of single chain forms of HF or PK may trigger-surface dependent reactions, but the vast majority of molecules are activated by reciprocal proteolysis between HF and PK with HMWK as a cofactor.

The mechanisms of hydrolysis of alkyl N-alkylthioncarbamate esters at 100 °C

2005 ◽  
Vol 83 (9) ◽  
pp. 1483-1491 ◽  
Author(s):  
Eduardo Humeres ◽  
Maria de Nazaré M. Sanchez ◽  
Conceição ML Lobato ◽  
Nito A Debacher ◽  
Eduardo P. de Souza
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Base Catalysis ◽  
Hydrolysis Rate ◽  
Negative Slope ◽  
General Base ◽  
Order Rate ◽  
Basic Hydrolysis ◽  
Rate Profile ◽  
Hydrolysis Of

The hydrolysis of ethyl N-ethylthioncarbamate (ETE) at 100 °C was studied in the range of 7 mol/L HCl to 4 mol/L NaOH. The pH–rate profile showed that the hydrolysis occurred through specific acid catalysis at pH < 2, spontaneous hydrolysis at pH 2–6.5, and specific basic catalysis at pH > 6.5. The Hammett acidity plot and the excess acidity plot against X were linear. The Bunnett–Olsen plot gave a negative slope indicating that the conjugate acid was less hydrated than the neutral substrate. It was concluded that the acid hydrolysis occurred by an A1 mechanism. The neutral species hydrolyzed with general base catalysis shown by the Brønsted plot with β = 0.48 ± 0.04. Water acted as a general base catalyst with (pseudo-)first-order rate constant, kN = 3.06 × 10–7 s–1. At pH > 6.5 the rate constants increased, reaching a plateau at high basicity. The basic hydrolysis rate constant of ethyl N,N-diethylthioncarbamate, which must react by a BAc2 mechanism, increased linearly at 1–3 mol/L NaOH with a second-order rate constant, k2 = 2.3 × 10–4 (mol/L)–1 s–1, which was 10 times slower than that expected for ETE. Experiments of ETE in 0.6 mol/L NaOH with an excess of ethylamine led to the formation of diethyl thiourea, presenting strong evidence that the basic hydrolysis occurred by the E1cb mechanism. In the rate-determining step, the E1cb mechanism involved the elimination of ethoxide ion from the thioncarbamate anion, producing an isothiocyanate intermediate that decomposed rapidly to form ethylamine, ethanol, and COS.Key words: alkylthioncarbamate esters, ethyl N-ethylthioncarbamate, ethyl N,N-diethylthioncarbamate, hydrolysis, mechanism.

Interactions of Heparin and a Covalently Linked Antithrombin Heparin Complex with the Components of the Fibrinolytic Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2211-2211
Author(s):  
Ankush Chander ◽  
Helen M Atkinson ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Serine Proteases ◽  
Conflicts Of Interest ◽  
Order Rate Constant ◽  
Second Order ◽  
Coagulation Cascade ◽  
Simultaneous Addition ◽  
Order Rate ◽  
Significant Difference

Abstract Abstract 2211 Introduction: Unfractionated heparin (UFH) is used for the prophylaxis and treatment of thromboembolic diseases. UFH catalyzes inhibition by antithrombin (AT) of the serine proteases in the coagulation cascade. Additionally, UFH has been shown to interact with components of the fibrinolytic pathway in vitro. However UFH has several limitations which impact its utility as a therapeutic agent. Our lab has developed a novel covalent antithrombin-heparin complex (ATH) which inhibits most serine proteases of the coagulation pathway significantly faster when compared to non covalent mixtures of AT and UFH. However, the interactions of ATH with the components of the fibrinolytic pathway have not been studied before. Thus, the present study investigates possible serpin-heparin interactions of AT + UFH vs ATH within the fibrinolytic pathway. Methods: Discontinuous second order rate constant assays under pseudo-first order conditions were carried out to obtain second order rate constant (k2) values for the inhibition of plasmin by AT+UFH versus ATH. Briefly, at specific time intervals 20 nM plasmin was inhibited by 200 nM AT + 0–5000 nM UFH or by 200 nM ATH in the presence of 2.5 mM Ca2+. Reactions were neutralized by the simultaneous addition of a solution containing polybrene and plasmin substrate S-2366™ in buffer. Residual plasmin activity was measured and the final k2 values calculated. For experiments involving tPA, wells containing 40nM tPA and increasing concentrations of AT, UFH or ATH, at mole ratios ranging from 0 to 20:1, were incubated for 15 min. Reactions with tPA were neutralized by simultaneous addition of a solution containing either polybrene and tPA substrate, S-2288™ in buffer, (ATH and UFH) or only the substrate S-2288™ in buffer (AT). Enzyme activity was then determined by measuring rate of substrate cleavage (Vmax). Results: When plasmin was inhibited by AT in the absence of UFH, k2 values of 2.82×105 +/− 4.46×104 M−1 min−1 were observed. The k2 values increased with addition of successively higher concentrations of UFH up to a plateau with maximal k2 of 5.74×106 +/− 2.78×105 M−1 min−1 at a UFH concentration of 3000nM. For inhibition of plasmin by ATH, k2 values of 6.39 × 106 +/− 5.88 × 105 M−1 min−1 were observed. Inhibition of plasmin by ATH was not significantly different when compared to the highest k2 values obtained with UFH. (p=0.36) No statistically significant difference in tPA enzyme activity was observed when Vmax values for tPA alone were compared with those in the presence of AT, UFH or ATH. (p=0.932, p=0.085, p=0.31 respectively) Significance: The characteristic shape of the curve obtained from the k2vs. UFH plot suggests that the mechanism responsible for inhibition of plasmin by AT+UFH involves conformational activation of the serpin. The k2 values in this study for inhibition of plasmin by both AT+UFH and ATH were three orders of magnitude lower than k2 values for inhibition of thrombin or factor Xa. Furthermore these results suggest that tPA is not inhibited by AT + UFH or ATH, and is not influenced by the presence of UFH alone. Cumulatively, this indicates that the fibrinolytic pathway is minimally impacted by AT + UFH or ATH, allowing maximal antithrombotic potential to be achieved during anticoagulation. Overall, the favourable anticoagulant properties of ATH combined with the findings of this study strengthens the utility of the covalent conjugate over conventional UFH for the treatment of thromboembolic disorders. Disclosures: No relevant conflicts of interest to declare.

Ambident nucleophiles. II. Nitrogen-bonded and carbon-bonded σ-complex formation in the reaction of pyrrolide anions with 1,3,5-trinitrobenzene

1982 ◽  
Vol 60 (15) ◽  
pp. 1988-1995 ◽  
Author(s):  
J. C. Halle ◽  
M. J. Pouet ◽  
M. P. Simonnin ◽  
F. Debleds ◽  
F. Terrier
Keyword(s):  
Aqueous Solution ◽  
Complex Formation ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Reactive Species ◽  
Second Order ◽  
Potassium Salts ◽  
Strong Base ◽  
Order Rate

Reaction of 1,3,5-trinitrobenzene (TNB) with pyrrole, 2,5-dimethyl pyrrole, and 2,4-dimethyl-3-ethyl pyrrole in the presence of a strong base (CH3O−) yields nitrogen- and/or carbon-bonded 1:1 σ-complexes in dimethylsulphoxide (DMSO). Depending on the stoichiometry of the reagents, 1:2 and 2:1 pyrrole–TNB diadducts are also formed. Identification of all complexes was effected by nmr. The reactive species are shown to be the pyrrolide ions and the results emphasize the ambident character of these anions towards an aromatic electrophile. Some of the complexes have been isolated as crystalline potassium salts when experiments are performed in acetonitrile. Among the isolable complexes, the kineticallybutnotthermodynamicallyfavored nitrogen adduct of pyrrole (5a) is remarkably unreactive. The second-order rate constant kH+ for is H+-catalyzed decomposition in aqueous solution is only 1 L mol−1 s−1 (t = 25 °C).

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