Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi - PubMed (original) (raw)
. 1996 Feb 27;35(8):2796-803.
doi: 10.1021/bi952322z.
Affiliations
- PMID: 8611586
- DOI: 10.1021/bi952322z
Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi
Y Hwang et al. Biochemistry. 1996.
Abstract
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.
Similar articles
- A mutation correcting the DNA interaction defects of a mutant phage lambda terminase, gpNu1 K35A terminase.
Hwang Y, Feiss M. Hwang Y, et al. Virology. 1999 Dec 20;265(2):196-205. doi: 10.1006/viro.1999.0055. Virology. 1999. PMID: 10600592 - Endonuclease and helicase activities of bacteriophage lambda terminase: changing nearby residue 515 restores activity to the gpA K497D mutant enzyme.
Hwang Y, Hang JQ, Neagle J, Duffy C, Feiss M. Hwang Y, et al. Virology. 2000 Nov 10;277(1):204-14. doi: 10.1006/viro.2000.0591. Virology. 2000. PMID: 11062051 - The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.
Catalano CE. Catalano CE. Cell Mol Life Sci. 2000 Jan 20;57(1):128-48. doi: 10.1007/s000180050503. Cell Mol Life Sci. 2000. PMID: 10949585 Free PMC article. Review.
Cited by
- Biophysical and structural characterization of a multifunctional viral genome packaging motor.
Prokhorov NS, Davis CR, Maruthi K, Yang Q, Sherman MB, Woodson M, White MA, Miller LM, Jarrold MF, Catalano CE, Morais MC. Prokhorov NS, et al. Nucleic Acids Res. 2024 Jan 25;52(2):831-843. doi: 10.1093/nar/gkad1135. Nucleic Acids Res. 2024. PMID: 38084901 Free PMC article. - Revolving ATPase motors as asymmetrical hexamers in translocating lengthy dsDNA via conformational changes and electrostatic interactions in phi29, T7, herpesvirus, mimivirus, E. coli, and Streptomyces.
Weitao T, Grandinetti G, Guo P. Weitao T, et al. Exploration (Beijing). 2023 Feb 5;3(2):20210056. doi: 10.1002/EXP.20210056. eCollection 2023 Apr. Exploration (Beijing). 2023. PMID: 37324034 Free PMC article. Review. - ATP serves as a nucleotide switch coupling the genome maturation and packaging motor complexes of a virus assembly machine.
Yang Q, Catalano CE. Yang Q, et al. Nucleic Acids Res. 2020 May 21;48(9):5006-5015. doi: 10.1093/nar/gkaa205. Nucleic Acids Res. 2020. PMID: 32255177 Free PMC article. - Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging.
delToro D, Ortiz D, Ordyan M, Sippy J, Oh CS, Keller N, Feiss M, Catalano CE, Smith DE. delToro D, et al. J Mol Biol. 2016 Jul 3;428(13):2709-29. doi: 10.1016/j.jmb.2016.04.029. Epub 2016 Apr 30. J Mol Biol. 2016. PMID: 27139643 Free PMC article. - Discovery of a new method for potent drug development using power function of stoichiometry of homomeric biocomplexes or biological nanomotors.
Pi F, Vieweger M, Zhao Z, Wang S, Guo P. Pi F, et al. Expert Opin Drug Deliv. 2016;13(1):23-36. doi: 10.1517/17425247.2015.1082544. Epub 2015 Aug 24. Expert Opin Drug Deliv. 2016. PMID: 26307193 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases