Real-time kinetics of the interaction between the two subunits, Escherichia coli thioredoxin and gene 5 protein of phage T7 DNA polymerase

J Biol Chem. 2003 Jun 13;278(24):21421-8. doi: 10.1074/jbc.M302310200. Epub 2003 Mar 31.


T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host. The holoenzyme is essential for the replication of the phage. We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance. Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling. Reduced thioredoxin bound g5p but oxidized thioredoxin did not. The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nm for thioredoxin with 4.7 x 104.M-1.s-1 and 10.5 x 10-5.s-1 as the corresponding association (ka) and dissociation (kd) rate constants. The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions. Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nm, whereas mutant W28A had a KD of 12.5 nm. No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A. The effect of temperature on KD and the changes in enthalpy (-DeltaH = 20.2 kcal.m-1) and entropy (TDeltaS =-8.4 kcal.m-1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.

MeSH terms

  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism*
  • Dose-Response Relationship, Drug
  • Entropy
  • Escherichia coli / metabolism*
  • Hot Temperature
  • Kinetics
  • Mutation
  • Protein Binding
  • Thermodynamics
  • Thioredoxins / chemistry
  • Thioredoxins / metabolism*
  • Time Factors
  • src Homology Domains


  • Thioredoxins
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase