Ac-Ala-Ala-OH

The crystal structure of a complex formed by the interaction between proteinase K and a designed octapeptide amide, N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2, has been determined at 2.5 A resolution and refined to an R-factor of 16.7% for 7,430 reflections in the resolution range of 8.0-2.50 A. The inhibitor forms a stable complex through a series of hydrogen bonds and hydrophobic interactions with the protein atoms and water molecules. The inhibitor is hydrolyzed between Phe4I and DAla5I (I indicates the inhibitor). The two fragments are separated by a distance of 3.2 A between the carbonyl carbon of Phe4I and the main-chain nitrogen of DAla5I. The N-terminal tetrapeptide occupies subsites S1-S5 (S5 for acetyl group), whereas the C-terminal part fits into S1'-S5' region (S5' for amide group). It is the first time that such an extended electron density for a designed synthetic peptide inhibitor has been observed in the prime region of an enzyme of the subtilisin family. In fact, the inhibitor fills the recognition site completely. There is only a slight rearrangement of the protein residues to accommodate the inhibitor. Superposition of the present octapeptide inhibitor on the hexapeptide inhibitor studied previously shows an overall homology of the two inhibitors, although the individual atoms are displaced significantly. It suggests the existence of a recognition site with flexible dimensions. Kinetic studies indicate an inhibition rate of 100% by this specifically designed peptide inhibitor.

The alpha,beta-unsaturated aromatic amino acids phenyldehydroalanine (PDA) and styryldehydroalanine (SDA) were synthesized and used as sensitive spectroscopic probes to study the acylation of papain by specific substrates and inhibitors. The spectral changes observed upon acylation of the enzyme with peptides containing these amino acids are large red shifts of the absorption maxima (lambda max) of the chromophores. The magnitudes of the absorption shifts were 49 nm (from 277 to 326 nm) for PDA peptide and 59 nm (from 318 to 377 nm) for SDA peptides. The following specific substrates were synthesized: Ac-Phe-Phe-PDA-OEt, Ac-Phe-PDA-NH2, Ala-Ala-Phe-SDA-OME, Ala-Ala-Phe-SDA-NH2, Lys-Ala-(o-benzyl)tyrosyl-SDA-OMe, and Lys-Ala-(o-benzyl)-tyrosyl-SDA-NH2. Similarly, the specific competitive inhibitors Ac-Phe-PDA (Ki = 5.3 X 10(-6) M), Z-Phe-SDA (Ki = 5.6 X 10(-5) M), Ala-Ala-Phe-SDA (Ki = 2.9 X 10(-5) M), and Lys-Ala-(o-benzyl)tyrosyl-SDA (Ki = 1.1 X 10(-5) M) were prepared. An additional chromophore was used to follow the noncovalent association of an inhibitor or substrate with papain, independently from the acylation or deacylation steps. This chromophore, which was introduced into the peptides at position P2, IS p-(p"-dimethylaminophenylazo) phenylalanine (DAP). The light absorption spectrum of DAP is dependent on its environment. The substrates Ala-Ala-DAP-SDA-OMe and Ala-Ala-DAP-SDA-NH2 and the competitive inhibitor Ala-Ala-DAP-SDA (Ki = 2.5 X 10(-6) M) were prepared. The noncovalent binding of these peptides to the active site of papain was followed either by the increase in the absorption at 480 nm or the decrease at 550 nm. With these petides the acylation and deacylation reactions could be followed simultaneously at 377 nm. The extent of acyl enzyme formation was found to decrease in a sigmoidal way with increasing pH, with a transition point around pH 5.5.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.