The crystal structure of the S642A mutant of mitochondrial
aconitase (mAc) with citrate bound has been determined
at 1.8 Å resolution and 100 K to capture this binding
mode of substrates to the native enzyme. The 2.0 Å
resolution, 100 K crystal structure of the S642A mutant
with isocitrate binding provides a control, showing that
the Ser → Ala replacement does not alter the binding
of substrates in the active site. The aconitase mechanism
requires that the intermediate product, cis-aconitate,
flip over by 180° about the Cα–Cβ double
bond. Only one of these two alternative modes of binding,
that of the isocitrate mode, has been previously visualized.
Now, however, the structure revealing the citrate mode
of binding provides direct support for the proposed enzyme
mechanism.