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Mechanistic enzymology of a methyltetrahydrofolate: Corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum of the acetyl-CoA pathway

Shaying Zhao, University of Nebraska - Lincoln

Abstract

A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N$\sp5$-methyl group from (6S)-methyltetrahydrofolate (CH$\sb3$-H$\sb4$folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP) in the acetyl-CoA pathway of anaerobic CO$\sb2$ fixation. This methyl transfer is similar to the first half reaction of cobalamin-dependent methionine synthase and a N$\sp5$-methyltetrahydromethanopterin:coenzyme M methyltransferase in methanogens. Studying the MeTr reaction is important to further our understanding of CO/CO$\sb2$ fixation under anaerobic conditions, and of one-carbon transfer reactions in eucaryotes and in methanogens. In order to elucidate the enzymatic mechanism of MeTr, presteady-state kinetics, steady-state kinetics, binding studies, spectroscopic and redox titration experiments were performed. Presteady-state data indicate that the Co(I)-C/Fe-SP performs a direct S$\rm\sb{N}$2 displacement of the methyl group of CH$\sb3$-H$\sb4$folate to form H$\sb4$folate and methyl-Co(III) without the intermediacy of Co(II). The kinetic mechanism involves a ternary complex in which substrates bind in a random order and independent of each other. Product inhibition experiments indicate a rapid equilibrium random Bi Bi mechanism suggesting substrate binding and product release are fast. The kinetic constants were: k$\rm\sb{cat}=13.5\pm0.9s\sp{-1}$ (at 25$\sp\circ$C), K$\rm\sb{m}$ for the C/Fe-SP = 69.0 $\pm$ 8.6 $\mu$M and K$\rm\sb{m}$ for (6S)-CH$\sb3$-H$\sb4$folate = 5.0 $\pm$ 0.6 $\mu$M. Methyl transfer to the C/Fe-SP is pH dependent. Both the forward and reverse reactions were fast at low pH with pK$\rm\sb{a}$'s ranging from 5.0 to 5.7. A different pH profile was obtained when free cobalamin was the methyl acceptor. The kinetic pK$\rm\sb{a}$ values closely matched the pK$\rm\sb{a}$ for the N$\sp5$ group of CH$\sb3$-H$\sb4$folate (pK$\rm\sb{a}$ = 5.0); however, the experimental evidence indicates that the pH dependence appears to result from a pH-dependent MeTr conformational change (pK$\rm\sb{a}$ = of 5.1). The conformational change is catalytically competent (k 40 s$\sp{-1}$) and partially rate-limiting. The hydrophobic interactions between MeTr and the C/Fe-SP appeared to increase the reduction potential of Co(II)/(I) couple by 50 mV and accelerate the reaction. A reaction scheme is proposed which involves two partially rate-limiting steps. At high pH, the conformational change and the interconversion of the ternary complexes (chemical step) control the rate and, at low pH, the interconversion of the ternary complexes primarily controls the rate. Substrate binding and product release are fast.

Subject Area

Biochemistry|Microbiology|Chemistry

Recommended Citation

Zhao, Shaying, "Mechanistic enzymology of a methyltetrahydrofolate: Corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum of the acetyl-CoA pathway" (1996). ETD collection for University of Nebraska-Lincoln. AAI9628258.
https://digitalcommons.unl.edu/dissertations/AAI9628258

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