Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.
The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc.
McGrath
W J
WJ
Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000, USA.
Baniecki
M L
ML
Peters
E
E
Green
D T
DT
Mangel
W F
WF
D000260:Adenoviruses, Human / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013223:Statistics as Topic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C091827:adenain; D003545:Cysteine