Interaction of the Adenovirus Proteinase with Protein Cofactors with High Negative Charge Densities

• Published in: Analytical and bioanalytical chemistry Vol. 44; no. 24; pp. 8721 - 8729
• Main Authors: Bajpayee, Neil S, McGrath, William J, Mangel, Walter F
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.06.2005
• Subjects: Adenovirus; Adenoviruses, Human - enzymology; Amino Acid Sequence; Computer Simulation; DNA, Viral - chemistry; DNA, Viral - metabolism; Kinetics; Models, Molecular; Peptide Fragments - chemistry; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; Protein Conformation; Viral Proteins - chemistry; Viral Proteins - metabolism
• ISSN: 0006-2960; 1618-2642; 1520-4995
• DOI: 10.1021/bi0502240

The interactions of the human adenovirus proteinase (AVP) with polymers with high negative charge densities were characterized. AVP utilizes two viral cofactors for maximal enzyme activity (k cat/K m), the 11-amino acid peptide from the C-terminus of virion precursor protein pVI (pVIc) and the viral DNA. The viral DNA stimulates covalent AVP−pVIc complexes (AVP−pVIc) as a polyanion with a high negative charge density. Here, the interactions of AVP−pVIc with different polymers with high negative charge densities, polymers of glutamic acid (polyE), were characterized. The rate of substrate hydrolysis by AVP−pVIc increased with increasing concentrations of polyE. At higher concentrations of polyE, the increase in the rate of substrate hydrolysis approached saturation. Although glutamic acid did not stimulate enzyme activity, glutamic acid and NaCl could displace DNA from AVP−pVIc·(DNA) complexes; the K i values were 230 and 329 nM, respectively. PolyE binds to the DNA binding site on AVP−pVIc as polyE and DNA compete for binding to AVP−pVIc. The equilibrium dissociation constant for 1.3 kDa polyE binding to AVP−pVIc was 56 nM. On average, one molecule of AVP−pVIc binds to 12 residues in polyE. Comparison of polyE and 12-mer single-stranded DNA interacting with AVP−pVIc revealed the binding constants are similar, as are the Michaelis−Menten constants for substrate hydrolysis. The number of ion pairs formed upon the binding of 1.3 kDa polyE to AVP−pVIc was 2, and the nonelectrostatic change in free energy upon binding was −6.5 kcal. These observations may be physiologically relevant as they infer that AVP may bind to proteins that have regions of negative charge density. This would restrict activation of the enzyme to the locus of the cofactor within the cell.