Fluorometric assay of turnip mosaic virus NIa protease

Abstract

Turnip mosaic virus (TuMV) NIa protease cleaves the viral polyprotein at seven distinct junctions out of nine. The amino acid sequences of the seven cleavage sites have three conserved amino acids, V, H, Q in positions P4, P2, P1, respectively. Small molecules as well as conjugated peptides were tested for proteolytic activity of the enzyme. None of small molecules tested, such as methylumbelliferyl-p-guanidinobenzoate, p-nitrophenyl-p'-guanidinobenzoate, p-nitrophenyl acetate, and methylumbelliferyl-N-acetylglutamate, were hydrolyzed, Ac-V-Y-H-Q-Mca was also not hydrolyzed, Intramolecularly quenched fluorogenic substrates Dns-P-V-Y-H-Q-A-W-NH2 and Dns-P-V-Y-H-Q-W-NH2 emitted fluorescence after addition of TuMV NIa protease. The proteolysis rate of Dns-P-V-Y-H-Q-A-W-NH2 was comparable to that of the tetradecapeptide with an optimum sequence, but Dns-P-V-Y-H-Q-WNH, was hydrolyzed at a slower rate, which was confirmed independently by HPLC analysis. These results suggest that intramolecularly quenched fluorogenic substrates can be used for the continuous assay of TuRMV NIa protease. (C) 2000 Academic Press.