We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K+ ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P′1, and V at P′2, at P′3 the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P6; basic amino acids at P5; and no clear tendency at P3. Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.

KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and its substrate specificity by employing a series of five substrates derived from the fluorescence resonance energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic residues with highest efficiency for R; furthermore, the S(3) to S(2)' subsites accepted most of the natural amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight peptide substrates were identified containing sequences of proteins found in testis and one with myelin basic protein sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides present in higher primates with broad antifungal and mucosal healing activities that are generated from the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclusively at R(25) (DSHAKRHHGYKRKFHEKHHSHRGYR(25)↓SNYLYDN) that is the first cleavage observed inside the salivary gland. In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs.

No abstract

Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln 53 and Glu 136 that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P 1 position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S 1 subsite of SGP. These results and the pH profiles of SGP activity showed that its S 1 subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp 57 and/or Asp 43 as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu 136 ) activated water, and establish a fundamental role of the S 1 subsite interactions in promoting catalysis.The recently established glutamic peptidase family (Family G1 in MEROPS, also known as the Eqolisins) is a novel group of acid peptidases having structures and mechanistic features distinct from the canonical peptidase families (1, 2), and so far, found only in the filamentous fungi (3) where they play key roles in fungal growth (4). Glutamic peptidases were first differentiated from aspartic peptidases as being insensitive to pepstatin and the absence of sequence similarity to the well characterized pepsin-like and retroviral aspartic peptidases (5, 6).The Scytalidoglutamic peptidase (SGP), 2 isolated from the wood-degrading fungus Scytalidium lignicolum, is the prototype of glutamic peptidases. Gln 53 and Glu 136 (in SGP numbering) were proposed to participate in the catalytic process because the single point mutants E136A, Q53A, and Q53E lost both the autoprocessing and the enzymatic activities (7). Sitedirected mutagenesis studies on Aspergillus niger glutamic peptidase established that these same residues constitute the catalytic dyad in this peptidase (8). The molecular structure of SGP was determined in its unbound native form, in complex with hydrolytic products and transition state peptide analogues (1, 9, 10). SGP has a unique ␤-strand tertiary structure among the known peptidases, and each layer of the sandwich is formed by seven antiparallel ␤-sheets. The catalytic residues Glu 136 and Gln 53 are both located in the same layer of the sandwich structure...