Characterization of Three Components of Human Adenovirus Proteinase Activity in Vitro

Walter F Mangel,Diana L Toledo,Mark T Brown,Jerri H Martin,William J Mcgrath

doi:10.1074/jbc.271.1.536

Walter F Mangel, Diana L Toledo + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.1.536

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Abstract

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.

Highlights

§ Science and Engineering Research Semester Program participant supported by the Office of Science Education and Technical Information, Department of Energy
The substrate (Leu-ArgGly-Gly-NH)2-rhodamine is cleaved to Leu-Arg-Gly-Gly-NHrhodamine by the adenovirus proteinase; this is accompanied by a 3500-fold increase in fluorescence that is proportional to the amount of proteinase
Little or no hydrolysis was observed with purified recombinant L3 23-kDa protein (recombinant endoproteinase) expressed in E. coli

Summary

EXPERIMENTAL PROCEDURES

Materials—Tris, HEPES, dithiothreitol, and 5,5Ј-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) were purchased from Sigma. The cysteine titration was done by adding 10 ␮l of pVIc stock solution to 0.99 ml of Ellman’s buffer (0.1 M NaH2PO4 (pH 7.3) and 1 mM EDTA containing 0.33 mM 5,5Ј-dithiobis(2-nitrobenzoic acid)) and monitoring the increase in absorbance at 412 nm. The lysate was clarified by centrifugation at 10,000 ϫ g for 10 min This fraction, named the supernatant, was loaded onto a 2.5 ϫ 25-cm TSK-DEAE column equilibrated in 50 mM Tris (pH 8.0), 15 mM NaCl, and 5 mM ␤-mercaptoethanol. The pellet was resuspended in 0.8 ml of 0.01 M HEPES (pH 8), 0.01 M octyl glucoside, and 0.001 M EDTA and centrifuged at 5000 ϫ g in a Centricon-30 until 90% of the liquid flowed through the membrane. Assays of fractions from each peak were performed in the presence of Ad2 DNA and rEP

RESULTS

DISCUSSION

Methods