scholarly journals The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain

1989 ◽  
Vol 259 (2) ◽  
pp. 443-452 ◽  
Author(s):  
D Kowlessur ◽  
M O'Driscoll ◽  
C M Topham ◽  
W Templeton ◽  
E W Thomas ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Ligand Binding ◽  
Transition State ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Binding Interactions ◽  
Electrostatic Fields ◽  
Transition State Geometry

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.

scholarly journals Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain

1989 ◽  
Vol 258 (3) ◽  
pp. 755-764 ◽  
Author(s):  
D Kowlessur ◽  
C M Topham ◽  
E W Thomas ◽  
M O'Driscoll ◽  
W Templeton ◽  
...  
Keyword(s):  
Transition State ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Catalytic Site ◽  
Amide Bond ◽  
Compound I ◽  
Binding Interactions ◽  
Transition State Geometry ◽  
S2 Subsite

1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2′-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2′-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2′-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

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 A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition

1990 ◽  
Vol 266 (3) ◽  
pp. 645-651 ◽  
Author(s):  
W Templeton ◽  
D Kowlessur ◽  
E W Thomas ◽  
C M Topham ◽  
K Brocklehurst
Keyword(s):  
Transition State ◽  
Binding Site ◽  
Kinetic Data ◽  
Model Building ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Structural Basis ◽  
Compound I

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and 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 of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence

1991 ◽  
Vol 275 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Y K Li ◽  
J Boggaram ◽  
L D Byers
Keyword(s):  
Isotope Effect ◽  
Rate Constant ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Acidic Group ◽  
Irreversible Inhibitors ◽  
Effects Of Ph ◽  
Solvent Isotope ◽  
Bell Shaped Curve

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

scholarly journals Ionization characteristics of the Cys-25/His-159 interactive system and of the modulatory group of papain: resolution of ambiguity by electronic perturbation of the quasi-2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe

1993 ◽  
Vol 290 (1) ◽  
pp. 289-296 ◽  
Author(s):  
G W Mellor ◽  
E W Thomas ◽  
C M Topham ◽  
K Brocklehurst
Keyword(s):  
Ph Dependence ◽  
Order Rate Constant ◽  
Ion Pair ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Interactive System ◽  
Data Set ◽  
Pka Value ◽  
Compound Ii ◽  
Pair State

1. A new thiol-specific reactivity probe 4,4′-dipyrimidyl disulphide [compound (VII), m.p. 110 degrees C, pKa of its monohydronated form 0.91] was synthesized and used to resolve the ambiguity of interpretation of the behaviour of papain (EC 3.4.22.2) in alkaline media known to depend to varying extents on two ionizations with pKa values approx. 8.0-8.5 and > or = 9.5 respectively. 2. A new extensive pH-second-order rate constant (k) data set for the reaction of papain with 2-(acetamido)-ethyl 2′-pyridyl disulphide (IV) demonstrated the existence of a striking rate maximum at pH approx. 4, the independence of k around pH 8 and the increase in k with increase in pH across a pKa value of 10.0, behaviour similar to that of other 2-pyridyl disulphides (R-S-S-2-Py) that lack key substrate-like binding sites in R. 3. Although the simplest interpretation of the pKa value of 10.0 assigns it to the formation of (Cys-25)-S-/(His-159)-Im from the ion-pair state of the papain catalytic site, another interpretation may be conceived in which this pKa value is assigned to another group remote from the catalytic site, the state of ionization of which modulates catalytic-site behaviour. This alternative assignment is shown to require compensating effects in the pH region around 8 such that the formation of (Cys-25)-S-/(His-159)-Im across pKa 8.0-8.5 is without net kinetic effect in the reactions of simple 2-pyridyl disulphides such as compound (IV) and 2,2′-dipyridyl disulphide (II). 4. The lower basicity of compound (VII) relative to that of compound (II) (pKa 2.45) was predicted to diminish or abolish the compensation postulated as a possibility in reactions of 2-pyridyl disulphides because of the decreased effectiveness of reaction via a (His-159)-Im+H-assisted transition state. The characteristics of the pH-dependence of the reaction of papain with compound (VII) which are quite different from those for its reaction with compound (II) support both this prediction and the alternative assignment with a value of 8.3 for the pKa of the formation of (Cys-25)-S-/(His-159)-Im. 5. Evidence that the behaviour of papain towards both substrates and some substrate-derived time-dependent inhibitors is determined not only by the loss of the (Cys-25)-S-/(His-159)-Im+H ion-pair state by dehydronation with pKa 8.3 but also by another ionization of pKa approx. 10.0 is briefly discussed.

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.

Oxidation of Nickel(II) and Manganese(II) by Peroxodisulfate in Aqueous Solution in the Presence of Molybdate. Crystal Structure of the (NH4)6[NiMo9O32].6H2O Product

1992 ◽  
Vol 45 (12) ◽  
pp. 1943 ◽  
Author(s):  
SJ Dunne ◽  
RC Burns ◽  
GA Lawrance
Keyword(s):  
Rate Constant ◽  
Linear Dependence ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
X Ray Diffraction ◽  
X Ray ◽  
First Order ◽  
Oxidant Concentration ◽  
Ph Range ◽  
Kinetics Of

Oxidation of Ni2+,aq, by S2O82- to nickel(IV) in the presence of molybdate ion, as in the analogous manganese system, involves the formation of the soluble heteropolymolybdate anion [MMogO32]2- (M = Ni, Mn ). The nickel(IV) product crystallized as (NH4)6 [NiMogO32].6H2O from the reaction mixture in the rhombohedra1 space group R3, a 15.922(1), c 12.406(1) � ; the structure was determined by X-ray diffraction methods, and refined to a residual of 0.025 for 1741 independent 'observed' reflections. The kinetics of the oxidation were examined at 80 C over the pH range 3.0-5.2; a linear dependence on [S2O82-] and a non-linear dependence on l/[H+] were observed. The influence of variation of the Ni/Mo ratio between 1:10 and 1:25 on the observed rate constant was very small at pH 4.5, a result supporting the view that the precursor exists as the known [NiMo6O24H6]4- or a close analogue in solution. The pH dependence of the observed rate constant at a fixed oxidant concentration (0.025 mol dm-3) fits dequately to the expression kobs = kH [H+]/(Ka+[H+]) where kH = 0.0013 dm3 mol-1 s-1 and Ka = 4-0x10-5. The first-order dependence on peroxodisulfate subsequently yields a second-order rate constant of 0.042 dm3 mol-1 s-1. Under analogous conditions, oxidation of manganese(II) occurs eightfold more slowly than oxidation of nickel(II), whereas oxidation of manganese(II) by peroxomonosulfuric acid is 16-fold faster than oxidation by peroxodisulfate under similar conditions.

The reaction of 2,4,6-trinitrobenzene sulfonic acid with pancreatic elastase

1970 ◽  
Vol 48 (11) ◽  
pp. 1249-1259 ◽  
Author(s):  
Leticia Rao ◽  
T. Hofmann
Keyword(s):  
Rate Constant ◽  
Sulfonic Acid ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Trinitrobenzene Sulfonic Acid ◽  
Amino Group ◽  
Tyrosine Residues ◽  
Inactivation Rate Constant ◽  
Lysine Residues ◽  
Ph Range

The reaction of elastase with trinitrobenzene sulfonic acid was investigated in the pH range 9–12. Elastase was found to be inactivated by 2,4,6-trinitrobenzene sulfonic acid. The pH dependence of the pseudo first-order inactivation rate constant showed a pK of 10.3 and gave a Hill plot coefficient of 1.15. Trinitrophenol did not inactivate the enzyme. These results indicate that the inactivation is due to the covalent reaction of trinitrobenzene sulfonic acid with a single group in the enzyme. This group is not the N-terminal since the loss of N-terminal valine was considerably slower than the loss of activity at pH 10.5. The inactivation of elastase with 2,4-dinitrofluorobenzene also showed no correlation with the loss of the N-terminal. When the enzyme was exhaustively treated and fully inactivated with trinitrobenzene sulfonic acid at pH 10.5, the N-terminal valine and two out of three lysine residues were trinitrophenylated. No evidence for the loss of histidine was found. One of the tyrosine residues may be trinitrophenylated as judged from the molar extinction of the trinitrophenylated protein, but it has not been possible to isolate a trinitrophenylated tyrosine-containing peptide. The results can be interpreted in one of two ways: (a) trinitrophenylation of a group with a pK of 10.3, not involved in the activity, inactivates because the introduction of the trinitrophenyl residue causes a denaturation of the enzyme; or (b) a group with a pK of 10.3 controls the active conformation of the enzyme. The results do not exclude the possibility that the N-terminal plays an important role in the activity of the enzyme. Below pH 10.5 the reactivity of the N-terminal is low, indicating that it is buried.At pH 9.0 only the ε-amino group of lysine in position 224 reacted with trinitrobenzene sulfonic acid and full activity was retained. The second-order rate constant for the trinitrophenylation of this group was 25 times higher than that of the ε-amino group of the α-N-benzoyllysine.

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 Visual Interpretation of the Meaning of kcat/KM in Enzyme Kinetics

2021 ◽  
Author(s):  
Chiwook Park
Keyword(s):  
Transition State ◽  
Enzyme Kinetics ◽  
Rate Constant ◽  
Reaction Rate ◽  
Reaction Pathway ◽  
Order Rate Constant ◽  
Reaction Energy ◽  
Energy Diagram ◽  
Order Rate ◽  
Main Challenge

kcat and kcat/KM are the two fundamental kinetic parameters in enzyme kinetics. kcat is the first-order rate constant that determines the reaction rate when the enzyme is fully occupied at a saturating concentration of the substrate. kcat/KM is the second-order rate constant that determines the reaction rate when the enzyme is mostly free at a very low concentration of the substrate. Both parameters provide critical information on how the enzyme lowers the energy barriers along the reaction pathway for catalysis. However, it is surprising how often kcat/KM is used inappropriately as a composite parameter derived by dividing kcat with KM to assess both catalytic power and affinity to the substrate of the enzyme. The main challenge in explaining the true meaning of kcat/KM is the difficulty to demonstrate how the reaction energetics of enzyme catalysis determines kcat/KM in a simple way. Here, I report a step-by-step demonstration on how to visualize the meaning of kcat/KM on the reaction energy diagram. By using the reciprocal form of the expression of kcat/KM with the elementary rate constants in kinetic models, I show that kcat/KM is a harmonic sum of several kinetic terms that correspond to the heights of the transition states relative to the free enzyme. Then, I demonstrate that the height of the highest transition state has the dominant influence on kcat/KM, i. e. the step with the highest transition state is the limiting step for kcat/KM. The visualization of the meaning of kcat/KM on the reaction energy diagram offers an intuitive way to understand all the known properties of kcat/KM, including the Haldane relationship.

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