MECHANISTIC STUDIES OF THE METHYLTRANSFERASE FROM CLOSTRIDIUM-THERMOACETICUM - ORIGIN OF THE PH-DEPENDENCE OF THE METHYL-GROUP TRANSFER FROM METHYLTETRAHYDROFOLATE TO THE CORRINOID IRON-SULFUR PROTEIN

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N-5 methyl group from (6S)-methyltetrahydrofolate (CH3- H(4)folate) 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-H(4)folate and either the C/Fe-SP or vitamin B-12 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 [C-14]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with (CO2)-C-14 [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 S(N)2 displacement of the methyl group of CH3-H(4)folate to form H(4)folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pK(a) similar to 5.5). Similar pH profiles were obtained by steady-state kinetics. The k(cat)/K-m values for the C/Fe-SP and CH3-H(4)folate in the forward direction and for the methylated C/Fe-SP and H(4)folate in the reverse direction increased as the pK was lowered (pK(a) similar to 5.3). A different pH profile was obtained with free cobalamin as the substrate; the k(cat)/K-m for CH3-H(4)folate and cobalamin (forward reaction) increased (pK(a) similar to 7.0) and the k(cat)/K-m for H(4)folate and methylcobalamin (reverse reaction) decreased (pK(a) similar to 5.3) as the pH was lowered. Thus, in the methylation of B-12, the rate-limiting step is different from that in the methylation of the C/Fe-SP. The kinetic pK(a) values closely matched the pK(a) for the N-5 group of CH3-H(4)folate (pK(a) = 5.1); however, the pH dependence of methyl transfer appears to result from ionization of a group on MeTr, not substrate. The ionization on MeTr results in a conformational change that is currently being studied.