The active site of enolase is located at the carboxlic end of the a/b barrel. Asp 246, glu 295, and asp 320 are the amino acids of the enzyme that are believed to interact with the conformational metal cation. The binding of a metal cation (shown in green) at the conformational site (site I) causes a conformational change, activating the enzyme and enabling 2-phosphoglycerate (2-PGA) to bind to the enzyme. Gln 167 and lys 396 help to neutralize and stabilize the carboxyl group of 2-PGA.

After 2-PGA is bound, the second metal cation can bind at the catalytic site of the enzyme (site II) to enable the catalytic activity. The exact location of this binding site has yet to be determined in x-ray crystallography studies.6 The loop from ser 36 to his 43 moves to enable the carbonyl of the backbone and the hydroxyl side chain of ser 39 to interact with the catalytic metal cation near the ligand binding site.7

 When the substrate is bound, its phosphate group is in contact with the imidazole sidechain of his 159 via hydrogen bonding. Upon catalytic activation, the val 153-phe 169 and ser 250-glu 277 loops move in a coordinated fashion to enable his 159 better access to the substrate in order to donate a proton to 2-PGA's phosphoryl group. The negative charge of the phosphoryl group is further neutralized by arg 374 and the catalytic metal ion cofactor. 6

Lys 345 then shifts closer to arg 374, causing it to lose its proton. This proton is simultaneously replaced by a proton from C-2 of 2-PGA. Finally, the proton that was hydrogen bonded to the hydroxyl group of 2-PGA jumps from glu 168 and glu 211 (who were sharing this proton) to the hydroxyl group of 2-PGA, which then breaks off and forms water. As a result, a double bond is formed between C-2 and C-3 of the ligand molecule, resulting in the formation of phosphoenolpyruvate (PEP).7 [ ](spin on/off)

MECHANICS OF THE REVERSE REACTION

In the reverse reaction, a water molecule is held by glu 168, glu 211 and his 373. This water molecule is deprotonated by a carboxylate group, and its remaining hydroxyl group is added to C-3 of PEP. The C-2/C-3 double bond is then eliminated by the donation of a proton from lys 345 to PEP's C-2, resulting in the formation of 2-PGA.7 This reaction is only performed if his-159 is not in proximity of the ligand.7 The water molecule needed for this reaction to occur is held by the enzyme in such a way that it blocks his 159's "access" to the substrate, thus enabling the reverse reaction to occur.