Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase.
Magnesium protoporphyrin IX methyltransferase (ChlM), an enzyme in the chlorophyll biosynthetic pathway, catalyses the transfer of a methyl group to magnesium protoporphyrin IX (MgP) to form magnesium protoporphyrin IX monomethyl ester (MgPME). S-Adenosyl-L-methionine is the other substrate, from which a methyl group is transferred to the propionate group on ring C of the porphyrin macrocycle. Stopped-flow techniques were used to characterize the binding of porphyrin substrate to ChlM from Synechocystis PCC6803 by monitoring tryptophan fluorescence quenching on a millisecond timescale. We concluded that a rapid binding step is preceded by a slower isomerization of the enzyme. Quenched-flow techniques have been employed to characterize subsequent partial reactions in the catalytic mechanism. A lag phase has been identified that has been attributed to the formation of an intermediate. Our results provide a greater understanding of this catalytic process which controls the relative concentrations of MgP and MgPME, both of which are implicated in signalling between the plastid and nucleus in plants.
Shepherd
Mark
M
Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. shepherd@secsg.uga.edu
Hunter
C Neil
CN
D002851:Chromatography, High Pressure Liquid; D007536:Isomerism; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008956:Models, Chemical; D011485:Protein Binding; D011524:Protoporphyrins / Q000096:biosynthesis* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D011524:Protoporphyrins; C039629:magnesium protoporphyrin; C033434:protoporphyrin IX monomethyl ester; D008780:Methyltransferases; C060234:magnesium-protoporphyrin methyltransferase