Characterization of three components of human adenovirus proteinase activity in vitro.
Human adenovirus contains a virion-associated proteinase activity essential for the development of infectious virus. Maximal proteinase activity in vitro had been shown to require three viral components: the L3 23-kDa protein, an 11-amino acid cofactor (pVIc), and the viral DNA. Here, we present a quantitative purification procedure for a recombinant L3 23-kDa protein (recombinant endoproteinase (rEP)) expressed in Escherichia coli and the procedure that led to the purification and identification of pVIc as a cofactor. The cofactors stimulate proteinase activity not by decreasing Km, which changes by no more than 2-fold, but by increasing kcat. rEP alone had a small amount of activity, the kcat of which increased 355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA as well. Curves of Vmax of rEP.pVIc complexes with the substrate (Leu-Arg-Gly-NH)2-rhodamine as a function of pH in the absence and presence of Ad2 DNA indicate that the pKa values of amino acids that affect catalysis are quite different from those that affect catalysis by the cysteine proteinase papain. The pKa values in the absence of Ad2 DNA are 5.2, 6.4, 6.9, 7.5, and 9.4, and those in its presence are 5.2, 6.5, 7.4, and 8.8.
Mangel
W F
WF
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Toledo
D L
DL
Brown
M T
MT
Martin
J H
JH
McGrath
W J
WJ
D000260:Adenoviruses, Human / Q000201:enzymology*; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C082284:L3 23K protein, adenovirus; C091827:adenain