Figure 3

Substrate channel cycle. (a) The nucleotide and carbon substrates (acetone and bicarbonate) travel to the binding site through the substrate channel that is observable in the ligand-free AC structure with an opening near the α/β subunit interface. (b) Binding of substrates and the subsequent  phosphorylation reactions presumably start the cycle by closing off the substrate channel, trapping nucleotide and possibly the two reactive intermediates inside, and opens a new channel within the α-subunit’s interior. This interior channel, which is easily identifiable in the AMP-bound structure, connects the site at which acetone and HCO₃ ⁻ are phosphorylated with the Mn active site 40 Å away. Glu89 shifts into a Mn-coordinating position when the interior channel opens. (c) The AMP bound structure, which now has a hexacoordinate Mn, represents the enzyme in the state encountered by the carboxy- and acetone-phosphates. (d) Our acetate/AMP bound structure describes two intermediates or possibly the acetoacetate product electrostatically displacing Glu89, which results in the formation of the Mn-proximal α-helix. This switch triggers the closing of the interior channel and the reopening of the substrate channel for nucleotide departure and new access. Acetoacetate may then exit through the dimer interface.