A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.

Abstract

The preferred amino acid residues at the P;and P2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P' or P2positions, where a set of 15 or 16 amino acids was introduced.Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termin, which allowed direct sequence analysis by We report here a rapid method for the quantitative determination of endoproteinase substrate specificity for subsites on the C-terminal (P')** side of the scissile bond of a peptide substrate that largely circumvents these limitations. This method'uses the proteinase-catalyzed competitive hydrolysis of defined peptide mixtures to map the subsite preferences of the proteinase. The' substrate is a mixture of N-acetylated peptides, identical in sequence except for a single position. The strategy is outlined in the example shown in Fig. 1 The exact determinants of 3C proteinase specificity are not well understood. In poliovirus, each of the eight 3C-specific polyprotein cleavage sites occurs between glutamine-glycine bonds; however, not all glutamine-glycine pairs in the polyprotein are cleaved, suggesting additional sequence or conformational determinants. In' other picornaviruses, the known or predicted polyprotein cleavage sites are not limited to glutamine-glycine pairs; the P' residues are replaced in some picornaviral polyproteins by serine, alanine, methionine, threonine, or valine (3,4

Peptoids: a modular approach to drug discovery.

“…Ac-WRY-OMe, a fragment of the proteinaceous inhibitor tendamistat, binds with a K, value of 100 ,uM (T. Guo and P.A.B., unpublished data); the corresponding peptoids show similar affinity, with the retro sequence 9 (K, 200 AuM) preferred slightly over the direct-translation peptoid 8 (K, 350 AM). In another example, both forward, 6, and retro, 5, peptoid analogs ofthe consensus substrate (Ac-ELATQSFS-NH2) of the hepatitis A virus 3C protease (23,38) were prepared, with the "conservative" substitution of Nhser for threonine. While the retro peptoid, 5 (K, 2 mM), competed with affinity comparable to that of the peptide substrate (Km 2.1 mM), the forward peptoid, 6, showed no inhibition at 10 mM concentration.…”

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B

“…The biological method involves the display of the peptide libraries on filamentous phage, the identification of the best substrates by molecular biology tools, and the cleavage site determination in each substrate [1][2][3][4]. Synthetic peptide libraries can contain millions of compounds, but the identification of the substrates and the cleavage site require robust analytical assays such as Edman degradation [5][6][7], mass spectrometry [8,9], and chromatography [10]. Support-bound fluorescence resonance energy transfer (FRET) 1 peptide libraries have been prepared by the process of split synthesis, resulting in a single peptide sequence on each of the resin beads [11].…”

Marked Differences between Metalloproteases Meprin A and B in Substrate and Peptide Bond Specificity

“…Determined by Peptide Libraries-Peptide libraries were used to identify preferred amino acids of substrates at or near the cleavage site and to map the primed subsite 2 binding region of meprins (25). A completely random dodecapeptide mixture acetylated at the amino terminus was partially digested with meprin A or B, and the digested peptides were subjected to amino-terminal peptide sequencing.…”