Probing the Catalytic Mechanism Involved in the Isocitrate Lyase Superfamily: Hybrid Quantum Mechanical/Molecular Mechanical Calculations on 2,3-Dimethylmalate Lyase

The
isocitrate lyase (ICL) superfamily catalyzes the cleavage of
the C(2)–C(3) bond of various α-hydroxy acid substrates.
Members of the family are found in bacteria, fungi, and plants and
include ICL itself, oxaloacetate hydrolase (OAH), 2-methylisocitrate
lyase (MICL), and (2R,3S)-dimethylmalate
lyase (DMML) among others. ICL and related targets have been the focus
of recent studies to treat bacterial and fungal infections, including
tuberculosis. The catalytic process by which this family achieves
C(2)–C(3) bond breaking is still not clear. Extensive structural
studies have been performed on this family, leading to a number of
plausible proposals for the catalytic mechanism. In this paper, we
have applied quantum mechanical/molecular mechanical (QM/MM) methods
to the most recently reported family member, DMML, to assess whether
any of the mechanistic proposals offers a clear energetic advantage
over the others. Our results suggest that Arg161 is the general base
in the reaction and Cys124 is the general acid, giving rise to a rate-determining
barrier of approximately 10 kcal/mol.