New substrates of papain, based on the conserved sequence of natural inhibitors of the cystatin family.
A series of peptide substrates with different fluorogenic leaving groups has been synthesized. The peptide moiety in these substrates mimics a highly conserved sequence (QVVAG) in the natural reversible inhibitors of cysteine proteinases, the cystatins, that participates to the tight binding of target proteinases. This sequence is invariably cleaved at the A-G bond when synthetic peptides containing it were incubated with papain. AEC and AMC fluorophores were therefore attached to the Ala residue to construct new substrates for cysteine proteinases. The solubility of the resulting substrates was improved by attaching a N-terminal gluconoyl group, or by introducing an arginyl residue at P5 (nomenclature of Schechter I, Berger A (1967) Biochem Biophys Res Commun 27, 157-162). Neither induced significant changes in the kcat/Km values with papain. Those values were all in the 10(5) M-1 s-1 range. The kcat/Km was increased 10-50-fold by using substrates with intramolecularly quenched fluorescence. With these, the enzyme specificity on both sides of the scissile bond can be investigated. The substrate Abz-QVVAGA-EDDnp is among the most sensitive papain substrates ever reported, with a kcat/Km value of 29 10(6) M-1 s-1. The positioning and conformation of the bound QVVA moiety within the active site of papain were predicted by molecular modelling using the X-ray coordinates of a peptide inhibitor-papain complex.
Serveau
C
C
Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334, Université François-Rabelais, Tours, France.
Juliano
L
L
Bernard
P
P
Moreau
T
T
Mayer
R
R
Gauthier
F
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D015891:Cystatins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D015891:Cystatins; D010455:Peptides; D010206:Papain