Kinetics of action of pepsin on fluorescent peptide substrates.

Abstract

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...

Structure and function of a serine carboxypeptidase adapted for degradation of the protein synthesis antibiotic microcin C7

“…This reflection for substrate preference through a change in k cat and not in K m is reminiscent of earlier work with serine proteases in which synthetic substrates designed with altered binding interactions at the P1′ and subsequent downstream binding subsites have led to nonsignificant changes in the K m for these enzymes, while displaying large variations in the k cat (15)(16)(17)(18). For MccF, the order of magnitude increase in k cat for DSA as compared to ESA can be attributed to the differential mode of stabilization of the acyl-enzyme transition state (formed after the departure of the adenine sulfonamide) in the active site.…”

Kinetics of Proteolytic Reactions in Nanoporous Materials

“…To analyze the fast reaction rates observed when using the enzyme-loaded MOSF, a modified sequential mechanism [29][30][31][32] was employed to model the proteolysis kinetics. In this mechanism, the enzyme combines with a substrate, such as a protein or a large peptide, at its active amino acid, and then cleaves it into two smaller peptides at a time.…”

“…Indeed, protease digestion involves a series of consecutive reactions cleaving the peptide chain at specific well-targeted points, such as arginine and lysine for trypsin. To illustrate this point, the classical sequential scheme, [29][30][31][32] which considers that the enzyme cleaves one protein or peptide at a time and that the generated peptide fragments diffuse away from the substrate, was employed and modified to simulate the proteolysis process. Digestion of myoglobin was employed here as an example to illustrate the high efficiency of the nanoconfined proteolysis.…”

Design of a Novel Antimicrobial Peptide Activated by Virulent Proteases

“…The kinetic values did not exceed our expectations, but were equivalent to that of pepsin degrading fluorescent peptides (28). In this study, we screened a peptide substrate optimized for the P 1 -P 3 specificity of Sap isozymes.…”