Isotope and Elemental Effects Indicate a Rate-Limiting Methyl Transfer as the
Initial Step in the Reaction Catalyzed by <i>Escherichia</i> <i>coli</i> Cyclopropane Fatty Acid
Synthase<sup>†</sup>
David F. Iwig
Anthony T. Grippe
Timothy A. McIntyre
Squire J. Booker
10.1021/bi048692h.s001
https://acs.figshare.com/articles/journal_contribution/Isotope_and_Elemental_Effects_Indicate_a_Rate_Limiting_Methyl_Transfer_as_the_Initial_Step_in_the_Reaction_Catalyzed_by_i_Escherichia_i_i_coli_i_Cyclopropane_Fatty_Acid_Synthase_sup_sup_/3319264
Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on
unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene
carbon of the cyclopropane ring derives from the activated methyl group of <i>S</i>-adenosyl-l-methionine
(AdoMet), affording <i>S</i>-adenosyl-l-homocysteine (AdoHcys) and a proton as the remaining products. This
reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated
and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and
mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes
have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic
analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) <i>Biochemistry</i> <i>43</i>,
http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, <i>Se</i>-adenosyl-l-selenomethionine (SeAdoMet) and <i>Te</i>-adenosyl-l-telluromethionine (TeAdoMet), and their intrinsic
reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the
electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet,
while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet >
SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA
synthase reaction, the kinetic parameters of the reaction, <i>k</i><sub>cat</sub> and <i>k</i><sub>cat</sub> <i>K</i><sub>M</sub><sup>-1</sup>, followed the series SeAdoMet
> AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton
loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of <i>S</i>-adenosyl-l-[<i>methyl</i>-<i>d<sub>3</sub></i>]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 ± 0.083,
supporting this conclusion and also indicating that the methyl transfer takes place via a tight s<sub>N</sub>2 transition
state.
2004-10-26 00:00:00
Escherichia coli Cyclopropane Fatty
methyl group
N 2 transition state
series SeAdoMet
methylene donor
proton loss
k cat K M
Elemental Effects Indicate
UFA
CFA synthase reaction
methyl transfer
TeAdoMet