Characterization of human adenovirus proteinase activity in disrupted virus particles.
Virus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 10(9) disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2-Rhodamine, the Km was 1.4 microM and the Vmax was 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKa similar to that of the active-site nucleophile of the cysteine proteinase papain. The curve of Vmax as a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis - one with a pKa of 6.2, another with a pKa of 7.2, and a third with a pKa of 8.3.
McGrath
W J
WJ
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Abola
A P
AP
Toledo
D L
DL
Brown
M T
MT
Mangel
W F
WF
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000260:Adenoviruses, Human / Q000201:enzymology*; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000494:pharmacology; D012235:Rhodamines; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity
D003902:Detergents; D004220:Disulfides; D011480:Protease Inhibitors; D012235:Rhodamines; D012997:Solvents; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D003546:Cysteine Endopeptidases; C091827:adenain