Kinetics of Malonyl-CoA Synthetase from Rhizobium trifolii and Evidences for Malonyl-AMP Formation as a Reaction Intermediate

摘要

The catalytic mechanism of malonyl-CoA synthetase from Rhizobium tifolii was investigated by the steady state kinetics and intermediate identification. Initial velocity studies and the product inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping-Pong Ter Ter system as the most probable steady state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 0.17【0.04 レM, 0.24【 0.18 レM and 0.045【0.026 レM for ATP, malonate and CoA, respectively. The TLC analysis of the 32P-labelled products in reaction mixture containing [ャ-32P] ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-NMR spectra of AMP product isolated from the 18O transfer experiment using [18O]malonate. Two resonances were observed, corresponding to AMP labelled with zero and one atom of 18O, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the TLC analysis of reaction mixture containing [メ -32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Ping-Pong Ter Ter mechanism deduced from the initial velocity and product inhibition studies.