Enolase does 2nd set up rxn: 2PG to PEP

Question 1

Enolase

Answer 1

homodimeric, several isozymes
complexes with 2 Mg’s before 2PG binds.

This means it is sequential ordered.

Question 2

Two different internal energies

Answer 2

There are two different internal energies involved in this rxn. One is for the substrate and the other is for the product.

You are redistributing where the energy is by changing the structure of your molecule, which makes more likely to donate phosphate to ADP.

(next step: pyruvate kinase takes the new molecule with changed structure, which has different free energy available to donate a phosphate)

Question 3

What is the rxn of 2PG to PEP characteristic of?

Answer 3

E1cB with a poor leaving group.

Question 4

How does the 3D positioning of active site of Enolase allow for the loss of a poor leaving group?

Answer 4

First, two Mg’s present help to offset 2-PG’s large negative charge.

Proton that must be removed, is expected to have a pKa of 30 (or higher due to the fact there is extra negative charge form adjacent phosphate as well as negative charge from adjacent carboxylic acid) . But somehow lysine is supposed to pull it off.

What to do?
Mg offsets the charge of the phosphate and carboxylate. This allows us to make a delocalized carbanion. Lys removes H. l.g. as OH gets protonated by Glu as it leaves.

Question 5

Rate determining step

Answer 5

loss of C3’s OH as H2O l.g
As OH is being lost, Glu protonates it.