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.

Studies on Horseradish Peroxidase. VII. A Kinetic Study of the Oxidation of Iodide by Horseradish Peroxidase Compound II

1971 ◽  
Vol 49 (18) ◽  
pp. 3059-3063 ◽  
Author(s):  
R. Roman ◽  
H. B. Dunford ◽  
M. Evett
Keyword(s):  
Ionic Strength ◽  
Horseradish Peroxidase ◽  
Kinetic Study ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Acid Dissociation ◽  
Ph Range ◽  
Compound Ii ◽  
Kinetics Of

The kinetics of the oxidation of iodide ion by horseradish peroxidase compound II have been studied as a function of pH at 25° and ionic strength of 0.11. The logarithm of the second-order rate constant decreases linearly from 2.3 × 105 to 0.1 M−1 s−1 with increasing pH over the pH range 2.7 to 9.0. The pH dependence of the reaction is explained in terms of an acid dissociation outside the pH range of the study.

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.

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

1990 ◽  
Vol 68 (6) ◽  
pp. 965-972 ◽  
Author(s):  
Yuchiong Hsuanyu ◽  
H. Brian Dunford
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Second Order ◽  
Prostaglandin H Synthase ◽  
Compound I ◽  
Stabilizing Agents ◽  
Order Rate ◽  
Ability To Act ◽  
Prostaglandin H ◽  
Compound Ii

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 °C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant K2,app, was found to fall within the range of 5.8 × 106 ± 0.4 × 106 M−1∙s−1 < K2,app < 1.8 × 107 ± 0.1 × 107 M−1∙s−1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme–substrate complex formation, with kcat = 22 ± 3 s−1, Km = 67 ± 10 μM, and the second-order rate constant k3,app = 2.0 × 105 ± 0.2 × 105 M−1∙s−1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 ± 0.5 s−1, Km = 49 ± 7 μM, and k3,app = 1.03 × 105 ± 0.07 × 105 M−1∙s−1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 ± 5 and 0.52 ± 0.05 s−1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 ± 5 and 0.33 ± 0.02 s−1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate. It is suggested that kinetic studies on this enzyme can be performed in the presence of diethyldithiocarbamate, if one is cognizant of the rate of reaction of the stabilizing agent with compounds I and II and corrects for it if necessary.Key words: prostaglandin H synthase, diethyldithiocarbamate, cyclooxygenase, peroxidase, stabilizing agents.

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.

Studies on Horseradish Peroxidase. XII. A Kinetic Study of the Oxidation of Sulfite and Nitrite by Compounds I and II

1973 ◽  
Vol 51 (4) ◽  
pp. 588-596 ◽  
Author(s):  
R. Roman ◽  
H. B. Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Ground State ◽  
Ph Dependence ◽  
Intermediate Formation ◽  
Ion Concentration ◽  
Compound I ◽  
Hydrogen Ion Concentration ◽  
Ph Range ◽  
Compound Ii ◽  
Kinetics Of

The kinetics of the oxidation of sulfite and nitrite by horseradish peroxidase compounds I and II have been studied as a function of pH at 25° and ionic strength 0.11. The pH dependence of the rate of the reaction between compound I and sulfite over the pH range 2–7 is interpreted in terms of two ground state enzyme dissociations with pka values of 5.1 and 3.3, and that for the compound II reaction with sulfite in terms of a single ground state enzyme dissociation with a pKa value of 3.9. Whereas the reaction between compound I and sulfite produces the native enzyme without the intermediate formation of compound II, the reaction of compound I with nitrite yields compound II. The second-order rate constants for the reactions of compounds I and II with nitrite increase linearly with increasing hydrogen ion concentration over the pH range 6–8.

THE KINETICS OF THE CHELATION OF NICKEL (II) AND 1,10-PHENANTHROLINE

1964 ◽  
Vol 42 (4) ◽  
pp. 934-940 ◽  
Author(s):  
P. F. Barrett ◽  
W. MacF. Smith
Keyword(s):  
Ionic Strength ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Second Order ◽  
Hydrogen Ion ◽  
Kinetic Behavior ◽  
Ion Concentrations ◽  
Order Rate ◽  
Kinetics Of ◽  
Limiting Values

The kinetics of the formation of the bidentate monocomplex of 1,10-phenanthroline and nickel (II) have been examined spectrophotometrically at ionic strength 0.5 over the range of temperatures 8 to 37 °C and over the range of hydrogen ion concentrations 0.01 to 0.30 molar. The kinetic behavior over the range of conditions is consistent with that found at 25 °C by Margerum, Bystroff, and Banks. The limiting values for the second-order rate constant for the reaction at high acidities have been assessed and imply associated values of ΔH≠and ΔS≠ of 9.5 kcal mol−1 and −5.3 e.u. respectively.

Effect of Thrombomodulin on the Molecular Recognition and Early Catalytic Events in Thrombin-Protein C Interaction

1996 ◽  
Vol 76 (04) ◽  
pp. 556-560 ◽  
Author(s):  
Raimondo De Cristofaro
Keyword(s):  
Molecular Recognition ◽  
Perturbation Method ◽  
Rate Constant ◽  
Protein C ◽  
Order Rate Constant ◽  
Second Order ◽  
Concentration Addition ◽  
Order Rate ◽  
C Complex ◽  
Kinetics Of

SummaryA viscosity perturbation method allowed to compute the second order rate constant, k±15 for the formation of thrombin-Protein C complex, both in the absence and presence of thrombomodulin (TM) at pH 8.00 and 37° C. In the absence of TM the second order rate constant was found equal to 7.9 ± 0.6 × 103 M-1 sec-1, whereas it was enhanced to 9.9 ± 0.4 × 104 M-1 s-1 by a saturating (100 nM) TM concentration. Addition of 5 mM Ca++ to solution containing 100 nM TM induced a further increase of k+1 value up to 7.3 ± 0.5 × 105 M-1 s-1. Moreover, it was demonstrated that the thrombin-PC complex undergoes the acy-lation reaction more rapidly than it dissociates to form free thrombin and substrate (stickiness ratio = 2.4 ± 0.9). This tendency is even favored when thrombin is bound to TM both in the absence and presence of Ca++ (stickiness ratio = 9 ± 6 in the absence of Ca++ and 16 ± 10 in the presence of Ca++). Altogether these results demonstrate that TM is able to positively affect both the molecular encounter and the kinetics of the early catalytic events of the thrombin-Protein C interaction.

scholarly journals Kinetics of the reaction of thrombin and α2-macroglobulin

1985 ◽  
Vol 231 (2) ◽  
pp. 417-423 ◽  
Author(s):  
R D Feinman ◽  
A I Yuan ◽  
S R Windwer ◽  
D Wang
Keyword(s):  
Rate Constant ◽  
Gel Electrophoresis ◽  
Bond Formation ◽  
Covalent Bond ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Second Order ◽  
Order Rate ◽  
Covalent Bond Formation ◽  
Kinetics Of

The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

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)

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