Acylation of α-Chymotrypsin by Oxygen and Sulfur Esters of Specific Substrates: Kinetic Evidence for a Tetrahedral Intermediate

Hirohara, Hideo; Bender, Myron L.; Stark, Richard S.

doi:10.1073/pnas.71.5.1643

Cited by 35 publications

(16 citation statements)

References 28 publications

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“…There is considerable evidence that the formation of the acyl enzyme proceeds via a tetrahedral adduct [14][15][16][17] formed by attack of the hydroxyl group of Ser-195 on the reactive carbonyl of the substrate. The conformation of this tetrahedral intermediate can be predicted by taking into account the steric restrictions imposed by the enzyme-substrate interactions and on the basis of the stereoelectronic theory recently proposed by Deslongchamps et al [18][19][20].…”

Section: Resultsmentioning

confidence: 99%

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

Bizzozero¹,

Zweifel²

1975

FEBS Letters

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Section: Resultsmentioning

confidence: 99%

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

Bizzozero¹,

Zweifel²

1975

FEBS Letters

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“…E, ES and EP are the free, Michaelis–Menten and acylated enzyme forms, whereas k 1 , k ‐1 , k 2 and k 3 are the kinetic constants for substrate binding, dissociation, acylation and deacylation, respectively. Within the overall acylation step of the canonical Scheme 1 ( k 2 ), tetrahedral intermediate (TI) formation ( k B ) or breakdown ( k A ) may be rate limiting [7,19–21] (Scheme 2).…”

Section: Methodsmentioning

confidence: 99%

The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pK_a value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na⁺ binding

et al. 2005

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Recently, a homozygous deletion mutation of one of the two contiguous Lys9 ⁄ Lys10 residues in the A-chain of a-thrombin (DK9) was identified in patients with severe prothrombin deficiency and hemorrhagic diathesis [1,2].Compared with the wild-type (WT) form, the specificity constants of hydrolysis by DK9 of the synthetic substrate d-Phe-Pip-Arg-pNA and fibrinopeptide A were found to be 18-and 60-fold lower, respectively. The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (DK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) to the wild-type (WT) and DK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pK a value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free DK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the DK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A-B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.Abbreviations a-NAPAP, N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine; Bis-Tris, (2-hydroxyethyl)iminotris(hydroxymethyl)methane;

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“…The results of studies on the inhibition of pepsin by substrate analogs (7) and of gel-filtration experiments on the binding of such analogs (8) were consistent with the conclusion that the values of Km estimated from steady-state kinetic measurements approximate the value of K,, the dissociation constant of the initial enzyme-substrate complex, and that the rate-limiting step in the over-all catalytic process occurs during the transformation of this complex. In the experiments described below, the validity of this conclusion was tested by means of stopped-flow fluorescence measurements of the kinetics of the cleavage of substrates of the type A-Phe-Phe-OP4P by porcine pepsin and Rhizopus-pepsin (9) has been applied to the study of the mechanism of the action of papain (11), chymotrypsin (10,12), and trypsin (13).…”

mentioning

confidence: 99%

Kinetics of action of pepsin on fluorescent peptide substrates.

Sachdev¹,

Fruton²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Oligopeptide substrates of porcine pepsin (E) of the type A-Phe-Phe-B (S) that are cleaved solely at the PhePhe bond under the conditions of these studies, and bearing an amino-terminal fluorescent probe group (mansyl or dansyl), have been used for stopped-flow measurements of the rate of formation of the A-Phe product. These experiments were conducted under conditions of [El >> [S], and the kinetic data were compared with those obtained under conditions of [S] >> [El for the formation of the Phe-B product (the same in all cases). The results for substrates with A = mansyl-Gly, mansyl-Gly-Gly, and dansyl-Gly-Gly support the conclusion that the rate-limiting step in the over-all catalytic process is associated with the scission of the Phe-Phe bond in the first detectable ES complex. Although the rate of this step varies widely with the nature of the A portion of A-Phe-Phe-B, the magnitude of the dissociation constant of ES is relatively invariant. This supports the view that, in the cleavage of oligopeptide substrates by pepsin, secondary enzyme-substrate interactions may cause conformational changes at the catalytic site, and that a portion of the total binding energy may be used for the attainment of the transition state in the bondbreaking step. With substrates that are hydrolyzed extremely rapidly (A = dansyl-Gly-Ala, dansyl-Ala-Ala), the rate of formation of the A-Phe product appears to be faster than the steady-state rate, suggesting that an additional step has become kinetically significant in the over-all process. This step may be associated with the return of the conformation of the active site to its original state. In previous studies from this laboratory (1-6), a series of synthetic oligopeptide substrates of the type A-Phe-Phe-OP4P was described for porcine pepsin and other acid proteinases. The pyridylpropyloxy (OP4P) (8) were consistent with the conclusion that the values of Km estimated from steady-state kinetic measurements approximate the value of K,, the dissociation constant of the initial enzyme-substrate complex, and that the rate-limiting step in the over-all catalytic process occurs during the transformation of this complex. In the experiments described below, the validity of this conclusion was tested by means of stopped-flow fluorescence measurements of the kinetics of the cleavage of substrates of the type A-Phe-Phe-OP4P by porcine pepsin and Rhizopus-pepsin (9) has been applied to the study of the mechanism of the action of papain (11), chymotrypsin (10, 12), and trypsin (13).In the present work, advantage was taken of the earlier finding (3, 4, 6) that the fluorescence of the dansyl (Dns) or mansyl (Mns) group of compounds such as Dns-(or Mns-) Gly-Gly-Phe-Phe-OP4P is greatly enhanced when they are bound by pepsin and that cleavage products (e.g., Dns-GlyGly-Phe) bearing the fluorescent probe group are bound much more weakly. Consequently, stopped-flow measurements of the rate of decrease of the fluorescence of the en- MATERIALS AND METHODSPorcine pepsin was prepared f...

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Acylation of α-Chymotrypsin by Oxygen and Sulfur Esters of Specific Substrates: Kinetic Evidence for a Tetrahedral Intermediate

Cited by 35 publications

References 28 publications

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pK_a value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na⁺ binding

Kinetics of action of pepsin on fluorescent peptide substrates.

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Acylation of α-Chymotrypsin by Oxygen and Sulfur Esters of Specific Substrates: Kinetic Evidence for a Tetrahedral Intermediate

Cited by 35 publications

References 28 publications

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

The importance of the conformation of the tetrahedral intermediate for the α‐chymotrypsin‐catalyzed hydrolysis of peptide substrates

The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding

Kinetics of action of pepsin on fluorescent peptide substrates.

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The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pK_a value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na⁺ binding