posted on 2015-11-19, 09:06authored byJanid Asghar. Ali
Purification of the DNA gyrase B protein consistently led to two contaminating bands of 47kDa and 43kDa molecular masses. These were found to be the C- and N-terminal fragments of GyrB. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA. This was due to about 90% of GyrB being in an uncoupled form. The uncoupled GyrB was found to have a relatively high ATPase activity, therefore an in-depth kinetic study on DNA gyrase was not possible. Kinetic studies were carried out on the 43kDa protein, which is a cloned N-terminal fragment of GyrB. The 43kDa protein was found to hydrolyse ATP at a relatively low rate, with 10 muM 43kDa having and apparent kcat of 0.01 s-1, and 20 muM a kcat of 0.02 s-1. A greater than first order dependence of rate upon 43kDa concentration was observed in the concentration range of 2-40 muM. Hyperbolic type kinetics were observed at constant 43kDa concentration (5, 10, 20 and 40 ?M) for the rate with respect to ATP concentration. A model which was found to be consistent with molecular weight studies and the kinetic data has been proposed. The 43kDa monomer can bind ATP but is not competent to hydrolyse ATP. Hydrolysis can only occur in the context of a 43kDa2ATP2 dimer, which leads to the collapse of the dimer into monomers and release of products. The rate limiting step at the protein concentration range used is the dimerisation step. Novobiocin and coumermycin inhibit the ATPase reaction, with novobiocin binding at a stoichiometry of 1 novobiocin molecule to 1 43kDa monomer and coumermycin binding with a stoichiometry of 1 molecule to two molecules of 43kDa protein. The inhibition by coumarin drugs appears non-competitive. ADPNP binding to the 43kDa protein was found to be slow, with a second order rate constant of 0.86 M-1s-1 to 9.9 M-1s-1. ADPNP seems to bind with stoichiometries varying from 2 per 43kDa dimer to 1 per 43kDa dimer. ATP and ADP inhibit the amount of ADPNP bound, with ATP having no effect on the rate of ADPNP binding and ADP decreasing the rate of ADPNP binding. Novobiocin and coumermycin inhibit ADPNP binding to the 43kDa protein. ADPNP dissociates from the 43kDa protein at a very slow rate, with a half life of about 8 days. ATP and ADP have little effect on this rate. However high concentrations of novobiocin (10-100 mM) dramatically increase the rate of ADPNP dissociation from the 43kDa protein, indicating different coumarin and nucleotide binding sites.