Fluorometric assay of turnip mosaic virus NIa protease

Turnip mosaic virus (TuMV) NIa protease cleaves the viral polyprotein at se ven distinct junctions out of nine. The amino acid sequences of the seven c leavage sites have three conserved amino acids, V, H, Q in positions P4, P2 , P1, respectively. Small molecules as well as conjugated peptides were tes ted for proteolytic activity of the enzyme. None of small molecules tested, such as methylumbelliferyl-p-guanidinobenzoate, p-nitrophenyl-p'-guanidino benzoate, p-nitrophenyl acetate, and methylumbelliferyl-N-acetylglutamate, were hydrolyzed, Ac-V-Y-H-Q-Mca was also not hydrolyzed, Intramolecularly q uenched fluorogenic substrates Dns-P-V-Y-H-Q-A-W-NH2 and Dns-P-V-Y-H-Q-W-NH 2 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 tetradecapepti de with an optimum sequence, but Dns-P-V-Y-H-Q-WNH, was hydrolyzed at a slo wer 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.