Detection of native peptides as potent inhibitors of enzymes

Abstract

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in a-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 · 10 )11 m and thus works as a highly potent competitive inhibitor. The commercially available a-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated a-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 · 0.15 · 0.10 mm 3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of a-chymotrypsin, which was readily interpreted as a 14 residue fragment of a-chymotrypsin corresponding to Ile-Val-Asn-Gly-GluGlu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 r between the active site Ser195 Oc and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oc and Trp29 C Abbreviations OW, water molecule;

Designing Photoswitchable Peptides Using the AsLOV2 Domain

“…Peptides regulate a variety of biological processes by acting as competitive inhibitors (Singh, et al, 2005), allosteric regulators (Lockless and Ranganathan, 1999) and localization signals (Conti, et al, 1998). Photocontrol of peptide activity is a powerful tool for precise spatial and temporal control of cellular function (Gautier, et al, 2010; Nguyen, et al, 2004).…”

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

“…These residues are structurally equivalent to Ser195 and Gly193, respectively, in chymotrypsin. Thus, the geometry of the catalytic triad and the oxyanion hole are well conserved among clan PA peptidases28293031 (Fig. 5).…”

Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

“…The 14 amino acid long peptide turned out to have affinity for the active site of the enzyme. The X-ray diffraction data showed the peptide bound to the active site and the crystal was catalytically inactive [15]. Nevertheless, these observations also pointed out that TPP treated enzyme had a different conformation which was more vulnerable to autolysis during the long period of crystallization.…”

“…We had observed that TPP treatment of alpha chymotrypsin leads to a higher activity for the enzyme in both aqueous and low water media [10]. Unfortunately, our attempts at obtaining the structure of TPP treated alpha chymotrypsin by X-ray diffraction did not succeed [15]. During crystallization attempts, TPP treated alpha chymotrypsin underwent a selective autocatalytic cleavage near the N-terminus.…”