Journal
BIOCHEMISTRY
Volume 40, Issue 48, Pages 14468-14474Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi011562d
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- NIAID NIH HHS [AI41599] Funding Source: Medline
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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 Cys 104 of AVP has formed a disulfide bond with conserved Cys 10 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 ofthe 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 muM. The Ki for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the Kd for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 /iM, 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.
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