Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase.
In the course of catalysis or signaling, large multimodular proteins often undergo conformational changes that reposition the modules with respect to one another. The mechanisms that direct the reorganization of modules in these proteins are of considerable importance, but distinguishing alternate conformations is a challenge. Cobalamin-dependent methionine synthase (MetH) is a 136-kDa multimodular enzyme with a cobalamin chromophore; the color of the cobalamin reflects the conformation of the protein. The enzyme contains four modules and catalyzes three different methyl transfer reactions that require different arrangements of these modules. Two of these methyl transfer reactions occur during turnover, when homocysteine is converted to methionine by using a methyl group derived from methyltetrahydrofolate. The third reaction is occasionally required for reactivation of the enzyme and uses S-adenosyl-L-methionine as the methyl donor. The absorbance properties of the cobalamin cofactor have been exploited to assign conformations of the protein and to probe the effect of ligands and mutations on the distribution of conformers. The results imply that the methylcobalamin form of MetH exists as an ensemble of interconverting conformational states. Differential binding of substrates or products alters the distribution of conformers. Furthermore, steric conflicts disfavor conformers that juxtapose a methyl group on substrate with one on methylcobalamin. These results suggest that the methylation state of the cobalamin will influence the distribution of conformers during turnover.
Bandarian
Vahe
V
Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Ludwig
Martha L
ML
Matthews
Rowena G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism; D008715:Methionine / Q000096:biosynthesis; D008745:Methylation; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D013763:Tetrahydrofolates / Q000378:metabolism; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000737:chemistry*
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013763:Tetrahydrofolates; D006710:Homocysteine; D012436:S-Adenosylmethionine; D008715:Methionine; C019476:mecobalamin; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12; C005984:5-methyltetrahydrofolate