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1.
Bajpayee NS  McGrath WJ  Mangel WF 《Biochemistry》2005,44(24):8721-8729
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.  相似文献   

2.
The interaction of the human adenovirus proteinase (AVP) and AVP-DNA complexes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 microM. The binding of AVP to 12-mer single-stranded DNA decreased the K(d) for the binding of pVIc to AVP to 0.09 microM. The pVIc-AVP complex hydrolyzed the substrate with a Michaelis constant (K(m)) of 3.7 microM and a catalytic rate constant (k(cat)) of 1.1 s(-1). In the presence of DNA, the K(m) increased less than 2-fold, and the k(cat) increased 3-fold. Alanine-scanning mutagenesis was performed to determine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Two amino acid residues, Gly1' and Phe11', were the major determinants in the binding of pVIc to AVP, while Val2' and Phe11' were the major determinants in stimulating enzyme activity. Binding of AVP to DNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP. Binding of either or both of the cofactors, pVIc or the viral DNA, to AVP did not dramatically alter its secondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably by prematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly, thereby aborting the infection.  相似文献   

3.
Human adenovirus proteinase (AVP) requires two cofactors for maximal activity: pVIc, a peptide derived from the C terminus of adenovirus precursor protein pVI, and the viral DNA. Synchrotron protein footprinting was used to map the solvent accessible cofactor binding sites and to identify conformational changes associated with the binding of cofactors to AVP. The binding of pVIc alone or pVIc and DNA together to AVP triggered significant conformational changes adjacent to the active site cleft sandwiched between the two AVP subdomains. In addition, upon binding of DNA to AVP, it was observed that specific residues on each of the two major subdomains were significantly protected from hydroxyl radicals. Based on the locations of these protected side-chain residues and conserved aromatic and positively charged residues within AVP, a three-dimensional model of DNA binding was constructed. The model indicated that DNA binding can alter the relative orientation of the two AVP domains leading to the partial activation of AVP by DNA. In addition, both pVIc and DNA may independently alter the active site conformation as well as drive it cooperatively to fully activate AVP.  相似文献   

4.
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.  相似文献   

5.
Brown MT  Mangel WF 《FEBS letters》2004,563(1-3):213-218
Actin bound to the adenovirus proteinase (AVP) with a lower equilibrium dissociation constant, 4.2 nM, than those exhibited by two viral, nuclear cofactors for AVP, the 11-amino acid peptide pVIc and the viral DNA. The k(cat)/K(m) ratio for substrate hydrolysis by AVP increased 150,000-fold in the presence of actin. The 11-amino acid residue peptide corresponding to the C-terminus of actin, which is highly homologous to pVIc, bound to AVP and stimulated its activity in the presence of DNA. As a cellular cofactor for AVP, AVP(actin) complexes may facilitate the cleavage of cytoskeletal proteins, preparing the infected cell for lysis and release of nascent virions.  相似文献   

6.
The adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is essential for the production of infectious virus, and here we report its structure at 0.98 Å resolution. AVP, initially synthesized as an inactive enzyme, requires two cofactors for maximal activity: pVIc, an 11-amino acid peptide, and the viral DNA. Comparison of the structure of AVP with that of an active form, the AVP-pVIc complex, reveals why AVP is inactive. Both forms have an α + β fold; the major structural differences between them lie in the β-sheet domain. In AVP-pVIc, the general base His-54 Nδ1 is 3.9 Å away from the Cys-122 Sγ, thereby rendering it nucleophilic. In AVP, however, His-54 Nδ1 is 7.0 Å away from Cys-122 Sγ, too far away to be able to abstract the proton from Cys-122. In AVP-pVIc, Tyr-84 forms a cation-π interaction with His-54 that should raise the pKa of His-54 and freeze the imidazole ring in the place optimal for forming an ion pair with Cys-122. In AVP, however, Tyr-84 is more than 11 Å away from its position in AVP-pVIc. Based on the structural differences between AVP and AVP-pVIc, we present a model that postulates that activation of AVP by pVIc occurs via a 62-amino acid-long activation pathway in which the binding of pVIc initiates contiguous conformational changes, analogous to falling dominos. There is a common pathway that branches into a pathway that leads to the repositioning of His-54 and another pathway that leads to the repositioning of Tyr-84.  相似文献   

7.
The crystal structure of the human adenovirus proteinase (AVP), a cysteine proteinase covalently bound to its 11-amino-acid peptide cofactor pVIc, has been solved to 1.6-A resolution with a crystallographic R-factor of 0.136, R(free)=0.179. The fold of AVP-pVIc is new and the structural basis for it is described in detail. The polypeptide chain of AVP folds into two domains. One domain contains a five-strand beta-sheet with two peripheral alpha-helices; this region represents the hydrophobic core of the protein. A second domain contains the N terminus, several C-terminal alpha-helices, and a small peripheral anti-parallel beta-sheet. The domains interact through an extended polar interface. pVIc spans the two domains like a strap, its C-terminal portion forming a sixth strand on the beta-sheet. The active site is in a long, deep groove located between the two domains. Portions are structurally similar to the active site of the prototypical cysteine proteinase papain, especially some of the Calpha backbone atoms (r.m.s. deviation of 0.354 A for 12 Calpha atoms). The active-site nucleophile of AVP, the conserved Cys(122), was shown to have a pK(a) of 4.5, close to the pK(a) of 3.0 for the nucleophile of papain, suggesting that a similar ion pair arrangement with His(54) may be present in AVP-pVIc. The interactions between AVP and pVIc include 24 non-beta-strand hydrogen bonds, six beta-strand hydrogen bonds and one covalent bond. Of the 204 amino acid residues in AVP, 33 are conserved among the many serotypes of adenovirus, and these aid in forming the active site groove, are involved in substrate specificity or interact between secondary structure elements.  相似文献   

8.
J Ding  W J McGrath  R M Sweet    W F Mangel 《The EMBO journal》1996,15(8):1778-1783
The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X-ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy.  相似文献   

9.
Hepatitis A virus (HAV) 3C proteinase is responsible for processing the viral precursor polyprotein into mature proteins. The substrate specificity of recombinant hepatitis A 3C proteinase was investigated using a series of synthetic peptides representing putative polyprotein junction sequences. Two peptides, corresponding to the viral polyprotein 2B/2C and 2C/3A junctions, were determined to be cleaved most efficiently by the viral 3C proteinase. The kcat/Km values determined for the hydrolysis of a further series of 2B/2C peptides, in which C-terminal and N-terminal amino acids were systematically removed, revealed that P4 through P2' amino acids were necessary for efficient substrate cleavage. The substitution of Ala for amino acids in P1 and P4 positions decreased the rate of peptide hydrolysis by 100- and 10-fold, respectively, indicating that the side chains of Gln in P1 and Leu in P4 are important determinants of substrate specificity. Rates of hydrolysis measured for other P1- and P4-substituted peptides indicate that S1 is very specific for the Gln side chain whereas S4 requires only that the amino acid in P4 be hydrophobic. A continuous fluorescence quench assay was developed, allowing the determination of kcat/Km dependence on pH. The pH rate profile suggests that catalyzed peptide hydrolysis is dependent on deprotonation of a reactive group having a pKa of 6.2 (+/- 0.2). The results of tests with several proteinase inhibitors indicate that this cysteine proteinase, like other picornaviral 3C proteinases, is not a member of the papain family.  相似文献   

10.
We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.  相似文献   

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