Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction.
Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. 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 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-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-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, 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 t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Kang
S W
SW
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D001426:Bacterial Proteins / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology*
D001426:Bacterial Proteins; D011756:Diphosphates; D008314:Malonates; D000249:Adenosine Monophosphate; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase