This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The adenovirus proteinase, an enzyme required for a successful adenovirus infection, is synthesized in an inactive form. It is activated by an 11-amino acid peptide, pVIc, and the viral DNA. This series of experiments has as its objective the elucidation of the mechanism by which the adenovirus proteinase is activated by pVIc. We have crystal structures of the active form of the enzyme, the AVP-pVIc complex (1.6 ? resolution) and the inactive form of the enzyme, AVP, (0.98 ? resolution). We have postulated that the active site nucleophile is Cys122, the general base is His54, and the oxyanion hole Gln115. AVP is inactive because His54 is too far away from Cys122 to abstract the proton from the sulfur. We have also postulated that Tyr84 forms a cation-pi interaction with His54 to lower the pKa of Cys122 and that Glu71 forms a hydrogen bond with His54 to freeze His54 in a position optimal to interact with the nucleophile. We have mutated all these amino acids to ala and are trying to obtain the crystal structures of the mutants with and without pVIc. These crystal structures should reveal how specific amino acids contribute to the catalytic mechanism. And those amino acids important to the mechanism of catalysis should be targets for antiviral drugs, the overall objective of our NIH grant.
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