Mechanism and Catalytic Site Atlas

Introduction

The initial enolisation step occurs with Glu 174 acting as a general base catalyst in concert with His 136 (stabilised by Asp 99) acting as a general acid catalyst. The C3 hydroxyl of the substrate is activated to become acidic by binding both metal ions and deprotonation may occur before or after the enolisation. The enolate ion is stabilised by the Mn ions, His 136, and Tyr 94. Collapse of the intermediate and dehydration is facilitated by Cys 66 acting as a general acid catalyst. An acid-base catalysed ketonisation process follows whereby the C2 hydroxyl is deprotonated by the His 136-Asp 99 dyad and the C1 is protonated by Glu 174. 1,2-skeleton rearrangement occurs by deprotonation of the C4 hydroxyl by Asp 41. A water activated by one magnesium ion acts as a nucleophile to hydrate the substrate and proton donation by His 136 yields the enolate form of 3,4-dihydroxy-2-butanone-4-phosphate. Loss of the formate results in a shift in coordination which allows a water molecule associated to both Mn ions to act as a proton donor in concert with Glu 174 acting as a general base catalyst to generate the final product.