Kinetic and hydrodynamic studies of the NodL O-acetyl transferase of Rhizobium leguminosarum: a random-order ternary complex mechanism for acetyl transfer by a roughly spherical trimeric protein.
The nodL gene product of Rhizobium leguminosarum is required for O-acetylation of diffusible lipo-oligosaccharide signalling factors which are involved in the host-specific nodulation of legume roots. Kinetic studies of the forward reaction, using the substrate analogues chitosan pentaose and chitosan tetraose and the acyl donors acetyl-CoA and propionyl-CoA, and the dead-end inhibitor EtCoA are consistent with a steady-state random-order ternary complex mechanism in which the off rate of the O-acetyl chitosan oligomer appears to be partially rate-determining. Moreover, the linearity of primary double-reciprocal plots favours the view that the interconversion of the ternary complex of NodL and its substrates with that of enzyme and bound products is not significantly faster than k(cat). Dissociation constants for coenzyme A and acetyl-CoA were determined by titration microcalorimetry to be 16.5 and 7.2 microM respectively, the latter in agreement with the kinetically derived value of 7.0 microM. The physical state of purified NodL, as determined by equilibrium centrifugation, velocity sedimentation and quasi-elastic light scattering, is that of a roughly spherical, trimeric protein with little tendency to self-associate.
Hindson
V J
VJ
Department of Biochemistry, University of Leicester, UK. vhindson@picr.man.ac.uk
Dunn
S O
SO
Rowe
A J
AJ
Shaw
W V
WV
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D007700:Kinetics; D008027:Light; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology*; D012542:Scattering, Radiation; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000123:Acetyltransferases