Mechanochemistry of t7 DNA helicase.
The bacteriophage T7 helicase is a ring-shaped hexameric motor protein that unwinds double-stranded DNA during DNA replication and recombination. To accomplish this it couples energy from the nucleotide hydrolysis cycle to translocate along one of the DNA strands. Here, we combine computational biology with new biochemical measurements to infer the following properties of the T7 helicase: (1) all hexameric subunits are catalytic; (2) the mechanical movement along the DNA strand is driven by the binding transition of nucleotide into the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind DNA; (4) the hydrolysis step changes the affinity of a subunit for DNA allowing passage of DNA from one subunit to the next. We construct a numerical optimization scheme to analyze transient and steady-state biochemical measurements to determine the rate constants for the hydrolysis cycle and determine the flux distribution through the reaction network. We find that, under physiological and experimental conditions, there is no dominant pathway; rather there is a distribution of pathways that varies with the ambient conditions. Our analysis methods provide a systematic procedure to study kinetic pathways of multi-subunit, multi-state cooperative enzymes.
Liao
Jung-Chi
JC
Departments of Molecular and Cell Biology and ESPM, University of California, Berkeley, CA 94720-3112, USA.
Jeong
Yong-Joo
YJ
Kim
Dong-Eun
DE
Patel
Smita S
SS
Oster
George
G
D000465:Algorithms; D017123:Bacteriophage T7 / Q000201:enzymology*; D002384:Catalysis; D019295:Computational Biology; D004247:DNA / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011995:Recombination, Genetic; D012984:Software; D013942:Thymine Nucleotides / Q000737:chemistry; D013997:Time Factors; D014867:Water / Q000737:chemistry
D004277:DNA, Single-Stranded; D013942:Thymine Nucleotides; D014867:Water; C041083:beta, gamma-methylenedeoxythymidine 5'-triphosphate; D004247:DNA; D019915:DNA Primase; D010100:Oxygen