Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.
Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.
Viney
Joanne
J
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom S10 2TN.
Davison
Paul A
PA
Hunter
C Neil
CN
Reid
James D
JD
D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008954:Models, Biological; D009994:Osmolar Concentration; D011485:Protein Binding; D011524:Protoporphyrins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology
D007477:Ions; D011524:Protoporphyrins; D011994:Recombinant Proteins; C028025:protoporphyrin IX; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium