A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.
We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate constant kcat/Km of 3.1 10(7) M-1S-1. It is also efficiently hydrolyzed by cathepsin L, although the kcat/Km for this proteinase is about 60-fold lower than that for papain. This change is due to a decrease in kcat, the Km's are almost identical. This allows clear functional discrimination between these two proteinases, and may lead to the development of selective inhibitors for individual cysteine proteinases. Unlike most commonly used papain substrates, Abz-QVVAGA-EDDnp is not hydrolyzed by trypsin. The papain cleavage site was identified as the A-G bond by N-terminal amino acid sequencing. The use of sensitive and specific substrates such as the one described here will prove invaluable for investigating cysteine proteinase activities in parasite infections. The close interaction between papain or cathepsin L with Abz-QVVAGA-EDDnp is compared to that with cystatin inhibitors, which all include a QxVxG consensus segment in their structure.
Gauthier
F
F
Laboratory of Enzymology and Protein Chemistry, CNRS URA 1334, University François Rabelais, Tours, France.
Moreau
T
T
Lalmanach
G
G
Brillard-Bourdet
M
M
Ferrer-Di Martino
M
M
Juliano
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000302:isolation & purification / Q000378:metabolism*; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity
D009842:Oligopeptides; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D010206:Papain