Mechanistic Studies of the Methyltransferase from Clostridium thermoaceticum: Origin of the pH Dependence of the Methyl Group Transfer from Methyl Tetrahydrofolate to the Corrinoid/Iron-Sulfur Protein

Abstract

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Cobalamin‐Dependent and Cobalamin‐Independent Methionine Synthases: Are There Two Solutions to the Same Chemical Problem?

“…The methyltransferase shows significant sequence similarity to residues 353 ± 623 of MetH [26], which constitute the CH 3 -H 4 folate-binding module of MetH. The pH-rate profiles obtained for the activities of CH 3 -H 4 folateÀcob(I)alamin and H 4 folateÀmethylcobalamin methyltransferase catalyzed by AcsE [27] are very similar to those shown for MetH in Fig. 4, so, the mechanism for this Me transfer is likely to be very similar, too.…”

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

“…Data for E. coli MetH show that protonation does not accompany formation of the binary complex with the folate substrate (35), but must occur in the ternary E⅐CH 3 -H 4 folate⅐cob(I)alamin complex. Curiously, there is no general acid in the neighborhood of N5, either in MetH or in related enzymes where N5 is protonated during the reaction, such as the homologous corrinoid͞iron-sulfur protein methyltransferase (AcsE) (38). The invariant Asn-508, which can interact with both O4 and N5 of the pterin ring (Fig.…”

Structural and Kinetic Evidence for an Extended Hydrogen-bonding Network in Catalysis of Methyl Group Transfer

“…, respectively (18). The experiment with the wild-type protein was performed with a DX.17MV sequential stopped-flow ASVD spectrophotometer from Applied Photophysics (Leatherbarrow, England), whereas for the N199A variant MeTr, the same experiments were performed at room temperature using an S2000 miniature fiber optic spectrometer (Ocean Optics, Inc., Dunedin, FL) inside a Vacuum Atmospheres (Hawthorne, CA) anaerobic chamber.…”