Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis.
Bacillus subtilis has three active tyrosine kinases, PtkA, PtkB and McsB, which play an important role in the physiology of the bacterium. Genome sequence analysis and biochemical experiments indicated that the ortholog of McsB, BAS0080, is the only active tyrosine kinase present in Bacillus anthracis. The autophosphorylation of McsB of B. anthracis was enhanced in the presence of an activator protein McsA (BAS0079), a property similar to that reported for B. subtilis. However, the process of enhanced phosphorylation of McsB in the presence of McsA remains elusive. To understand the activation mechanism of McsB, we carried out spectroscopic and calorimetric experiments with McsB and McsA. The spectroscopic results suggest that the binding affinity of Mg-ATP for McsB increased by one order from 10(3) to 10(4) in the presence of McsA. The calorimetric experiments revealed that the interaction between McsB and McsA is endothermic in nature, with unfavourable positive enthalpy (DeltaH) and favourable entropy (DeltaS) changes leading to an overall favourable free energy change (DeltaG). Kinetics of binding of both ATP and McsA with McsB showed low association rates (k(a)) and fast dissociation rates (k(d)). These results suggest that enhanced phosphorylation of McsB in the presence of McsA is due to increased affinity of ATP for McsB.
Mattoo
Abid R
AR
Institute of Genomics and Integrative Biology, Delhi, India.
Arora
Amit
A
Maiti
Souvik
S
Singh
Yogendra
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D001408:Bacillus anthracis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D004789:Enzyme Activation; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; C423321:McsA protein, Bacillus subtilis; D011994:Recombinant Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C423322:McsB protein, Bacillus subtilis; D011505:Protein-Tyrosine Kinases