Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.
Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.
Hwang
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Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Catalano
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Feiss
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation
D000345:Affinity Labels; D001386:Azides; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases