Detection of S1-P1 and S3-P3 interactions between papain and four synthetic substrates

In this study, the S1 - P1 and S3 - P3 interactions between papain and four synthetic peptide substrates were found as to be important. The values of Km were estimated as to be practically identical between these substrates; this latter is supporting the conclusions obtained by considering the estimated values of other kinetic parameters. Nevertheless, based on the estimated kcat and/or k cat/Km parameters of the used substrates, we concluded that an aromatic ring at the P3 position, and a positively charged side chain of the residue at the P1 position of the synthetic substrates were favored considerably their interaction with papain.<br>Neste estudo, o S1 - P1 e S3 - P3, interações entre papaina e quatro substratos sintéticos de pepetídios foram considerados importantes. Os valores de Km foram estimados e são praticamente idênticos entre esses substratos; Isso dá suporte as conclusões obtidas, considerando os valores parâmetros cinéticos estimados. No obstante, baseou na estimação parâmetros kcat e/ou k cat /Km dos substratos utilizados. Se pode concluir que um anel aromático na posição P3, e uma corrente carregada positivamente da cadeia do resíduo na posição P1 dos substratos sintéticos favoreceram interação com a papaina

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10(7) M(-1) sec(-1) range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10(5) M(-1) sec(-1) range due to lower k(cat) values, with the K-m's being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending an their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based an these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN, was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k(+0) = 1,100,000 M(-1) sec(-1)) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.UNIV TOURS,FAC MED,CNRS URA 1334,LAB ENZYMOL & CHIM PROT,F-37032 TOURS,FRANCEESCOLA PAULISTA MED,INFAR,DEPT BIOFIS,BR-04044020 São Paulo,BRAZILCNRS,CTR BIOPHYS MOLEC,LAB BIOCHIM GLYCOCONJUGUES & LECTINES ENDOGENES,F-45071 ORLEANS 2,FRANCEESCOLA PAULISTA MED,INFAR,DEPT BIOFIS,BR-04044020 São Paulo,BRAZILWeb of Scienc