A conformational change in the methyltransferase from Clostridium thermoaceticum facilitates the methyl transfer from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein in the Acetyl-CoA pathway.
The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron-sulfur protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate). This is an important reaction in the reductive acetyl-CoA pathway. The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the protein [Zhao, S., Roberts, D. L., & Ragsdale, S. W. (1995) Biochemistry 34, 15075-15083]. In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change. As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pK(a) = 5.4) and the emission intensity increased (pK(a) = 5.1). The extrinsic fluorescence probe, 4,4'-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change. The bis-ANS fluorescence was strongly enhanced upon binding MeTr. As the pH was decreased, the fluorescence was further enhanced and the emission maximum underwent a 14 nm blue shift (pK(a) = 5.0). By stopped-flow fluorescence studies, it was shown that these fluorescence changes occur at rates similar to the k(cat) for the MeTr reaction and thus reflect catalytically competent events. The combined results indicate that CH3-H4folate binds to a hydrophobic region in MeTr that includes a tryptophan residue(s). MeTr undergoes a pH-dependent conformational change that exposes this region to solvent and facilitates substrate binding.
Zhao
S
S
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln 68588-0664, USA.
Ragsdale
S W
SW
D000105:Acetyl Coenzyme A / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates; D003013:Clostridium / Q000737:chemistry*; D045728:Corrinoids; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013763:Tetrahydrofolates / Q000737:chemistry* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D045728:Corrinoids; D005456:Fluorescent Dyes; D007506:Iron-Sulfur Proteins; D011166:Porphyrins; D013763:Tetrahydrofolates; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000105:Acetyl Coenzyme A