title: Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase.
abstract: 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.
authors.affiliation: 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
authors.forename: Mark
authors.initials: M
authors.lastname: Shepherd
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
mesh_terms: 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
chemical_list: D011524:Protoporphyrins; C039629:magnesium protoporphyrin; C033434:protoporphyrin IX monomethyl ester; D008780:Methyltransferases; C060234:magnesium-protoporphyrin methyltransferase