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

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

Cofactors/Cosubstrates

Biotin is required and is covalently bound to the enzyme
A Mn²⁺ ion is also required for initiation of the aldol reaction.
CO₂ and ATP are cosubstrates, while ADP and PO₄⁼ are coproducts

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 a reversible reaction with water.
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).

Notes on this mechanism. This is an extremely compelx enzyme.... even beyound the obvious that it combines ATP hydrolysis and an aldol reaction. The individual reactions occur in separate places in the enzyme. There is one active site for the generation of carboxybiotin and a separate active site where the carboxul group is tranferred to pyruvate. This necessitates a "transport" of the biotin from site to site. Indeed the biotin is covalently attached to a third spot (the bioton carrier domain) whose sole function is to move the biotin from site to site. It is even more complex than this... The active enzyme is a tetramer (four identical subunits) which we call a, b, c and d. The biotin carboxylation site and the pyruvate carboxylation site are on one polypeptide (b for instance) while the biotin is supplied from another subunit (a in this case). See pictures below.

Furthermore, the human enzyme needs to be "activated" in order to function. The essential Mn²⁺ ion is held in place in the enzyme by several amino acids. One of these is a modified lysine. As you might guess a positively charged lys is not a very good thing for holding onto a positively charged metal atom. This lysine must first be carbolylated in order to be able to hold onto the metal ion that initiates the aldol reaction. This is likely done by biotin.

ΔG^o'

+3.1 kJ/M

K_eq

Comments

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.

Mechanism for Chemistry

Mechanism for Enzyme

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 (phosphate+carbonate) 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 and this therefore also a good "leaving group")

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 great leaving groups simply because they are compounds that are stable with all the charges etc at pH 7 JUST as they leave the reaction.... in this regard HO^- is not a grood leaving group because it would MUCH rather be protonated and neutral at pH 7.

Hover your mouse over a step to reveal a description

Pyruvate Carboxylase. Animation of the Pyruvate Caroxylase reaction Blue: represents the enzyme. "Start" begins an animation of the group transfer/ aldol reaction. It proceeds through the reaction in the "forward" direction and then "backwards" again. Note how the enzyme is involved. "+" increases speed while "-" decreases the animation speed. You may also step through the reaction using "next" or "previous"

This reaction occurs in several steps. in the direction of Gluconeogenesis... it starts with the "activation" of bicarbonate with ATP. bicarbonate attacks ATP for a group tranfer reaction. the intermediate is a phospho-carbonate anhydride. This is then attacked by the covalently bound Biotin cofactor to generate a carboxybiotin intermediate in a second group transfer reaction. The carboxybiotin is stable for quite some time. At some time later... a pyruvate enters the active site and in an Aldol reaction adds the carboxyl group to become oxaloacetate.

Compare the animated reaction to the "arrow pushing" scheme at the right. See if you can correlate the electron movement in the animation to the arrows in the static picture above.

Picture of Enzyme with substrate