Mechanism and Catalytic Site Atlas

Introduction

In this proposal the Asp 17 is no longer taking part in the catalytic mechanism, instead Glu 96 takes its role as general acid/base. Firstly Glu96 actS as a general base and deprotonates the conserved Lys132 in TalB via the catalytic water molecule to facilitate the nucleophilic attack of the NH2 group on the carbonyl carbon atom of Fructose 6-phosphate. The nucleophilic attack is followed by a protonation of the di-polar alkoxide in which the NH2R+ group serves as a proton source. Next, the Glu–H2O dyad acts as a general acid in the protonation of the C2-OH group of the neutral carbinolamine intermediate. Finally, a water molecule is released and the Schiff base intermediate is formed. Then Glu 96 deprotonates a water so that it can act as a base which will deprotonate the C4-OH and results in the heterolytic cleavage and the release of D-glyceraldehyde 3-phosphate. The DHA moiety is still bound to the lysyl residue as a carbanion which will undergo an isomerisation in which an electron pair will be transferred to the Nitrogen of Lys 132. The Lysine bound intermediate attacks the carbonyl carbon of D-erythrose 4-phosphate and the oxyanion formed will deprotonate Glu 96. Glu 96 then deprotonates a water which will attack the carabamylated carbon of the intermediate. Lastly Lys 132 deprotonates a bound waterwhich will initiate an elimination and result in the release of D-sedoheptulose 7-phosphate product from the enzyme. The active site is regenerated via protonation of Lys 132 by a bound water which in turn will accept a proton from Glu 96.