Pyruvate Carboxylase Information

Enzyme Name

Pyruvate Carboxylase

Reaction Catalyzed

Addition of CO₂ to C₃ of pyruvate via an Aldol Reaction

Reaction Type

Pyruvate adds "C" via an Aldol Reaction. There are two group transfer reactions to prepare CO₂ for this process

Rationale

This is the first of two required enzyme to bypass pyruvate kinase. This one puts another carboxyl group on to pyruvate... so that the next one, phosphoenolpyruvate carboxykinase, can use the Free Energy of decarboxylation to drive the addition of phosphate to the "enol" group.

Carbon dioxide cannot be used as CO₂ in mammalian systems for addition of a carbon by an Aldol reaction. First and foremost this is becasue we go to great lengths to keep our CO₂ concentration low. There are two mechanisms:

1. In the tissues, where CO₂ is generated and therefore in relatively high concentration, an enzyme called Carbonic Anhydrase (CA) rapdidly converts CO₂ to HCO₃^- + H⁺ by reaction with water in a reversible reaction.
2. In the lungs, where CO₂ concentration is low (atmosphereic concetrations), the same reaction catalyzed by the same enzyme runs the other way to generate CO₂ so that we can exhale it.

The first two steps in this overall reaction are solely to prepare and capture HCO₃^- so that it is amenable to an aldol reaction with pyruvate. For chemical reasons (see comments below) bicabonate is a very poor substrate for an aldol reaction.

The first preparation step is to "activate" HC0₃^- in a group transfer reaction with ATP. This generates the first intermediate, a carboxyl-phosphoanhydride. (notice the similarities of this to the phosphoanhydrides of ATP) and the first product, ADP.

The second capture step is to transfer the activated carbon dioxide to Biotin a cofactor that is covalently bound to the enzyme to generate carboxybiotin and phsophate. Carboxybiotin is kinetically stable and can now wait until pyruvate enters the active site.

The Aldol reaction between pyruvate and carboxybiotin generates the final product oxaloacetate as well as biotin (so that the enzyme can go through another cycle).

Pathway Involvement

Gluconeogenesis ONLY

This enzyme is the first of two that are required to bypass the pyruvate kinase reaction of glycolysis. Note that pyruvate kinase has a ΔG^o' that so favors glycolysis that it is unlikely that is could be driven backwards. Therefore two ATP (or ATP equivalent) dependent reactions: 1. pyruvate carboxylase 2. PEP carboxykinase, are required to get from pyruvate to phosphoenolpyruvate.

Cofactors/Cosubstrates

Biotin is required and is covalently bound to the enzyme

DG^o'

+3.1 kJ/M

Starting from standard state and allowing the reaction to come to equilibrium the oxaloacetate concentration would end up ~3 times lower than the concetration of pyruvate.

The Standard Free Energy slightly favors pyruvate production.

K_eq

Comments

In terms that a chemist would use... activation means adding a good "leaving group".
There are already four bonds to Carbon of bicarbonate... in order to "add" a C-C bond (aldol reaction) SOMETHING must leave bicarbonate. if bicarbonate were to react directly, HO^- would have to leave. At pH ~7 this is not very good at leaving as is becasue it is not very stable at this pH (is would much rather pick up a H⁺). We can make this work better, though, with ATP.

If bicarbonate first attacks ATP... then the "leaving group" is ADP. A very stable compound and is an excellent leaving group. and the end result is that mixed anhydride.

The next step is to have biotin attack the mixed anhydride where phosphate is the leaving group (phosphate is also very stable as is)

the last step is to have pyruvate do the aldo reaction with carboxybiotin.. now biotin is the leaving group as CO₂ is added. biotin is also a great leaving group..

What makes a "good" leaving group? a compound that is very stable as is... as it "leaves" the reactants. In these cases ADP, PO₄ and biotin all make greta leaving groups simply because they are compounds that are stable with all the charges etc at pH 7 JUST qas they leave the reaction.... n this regard HO^- is not a grood leaving group because it would MUCH rather be protonated and neutral at pH 7.

Mechanism for Chemistry

Mechanism for Enzyme

Picture of Enzyme with substrate

1. Ribbonspyrcarase. Here only the main chain is represented by these ribbons. There are four identical subunit
2. one subunit with substrate Three of the subunit deleted and a substrate analog (mannitol-1,6-bisphosphate) is added in the atom colored spheres. C=Gray; O=red; P=Orange. three critical AA in the active site are also added.
3. as above closeupSame picture as in "2" but zoomed in
4. substrate rotationas in "3" ribbons deleted and allowed to rotate to show the orientaion of these 3 amino acids. A.) LYS near C2 - this will form the Schiff's base; B.) GLU near C2 - will help the Schiff's base form by "attracting the water" as the Schiff's base forms; C.) LYS near C4 - will help the hydroxyl group deprotonate as the pyrcar reaction begins.
5. substrate and all nearby AAAll amino acid sidechains near the mannital are turn on
6. Schiff's base LYS in cyan and near GLU in yellow The LYS that will form the Schiff's base is color cyan, C2 of mannitol is colored magenta and the GLU is color yellow. The roles are highlighted in "4"
7. LYS near and near C_{4/sub> in cyan The LYS near C4 is colored cyan, C4 of mannitol is color magenta.}