Pyridoxal Phosphate I

Oxidation and Reduction

Redox reactions are characterized by a loss or gain of electrons. They can be looked at more easily however as a loss or gain of hydrogens. In the typical oxidation reaction below, the secondary alcohol has been oxidized to a ketone through the loss of 2 hydrogens. In the reduction reaction below, the disulfide is reduced to 2 thiols through the gain of 2 hydrogens. Oxidation reactions are exothermic and create energy while reduction reactions are endothermic and require energy to occur.

Download the chemdraw file above

The human body has evolved to maximize energy transfer. When an endothermic reaction must occur, it would be far to costly to create energy in order to power such a reaction. Therefore, endothermic and exothermic reactions are coupled together to minimize wasted energy.  The perfect way to minimize energy loss in biological systems would be to couple oxidation and reduction reactions (Redox reactions). So what does the body use to couple with oxidation and reduction reactions? We've seen how proteins are used to catalyze other reactions like hydrolysis, but amino acids are not good for catalyzing redox reactions. There are two apparent problems that do not allow proteins to easily undergo redox reactions, both involving the amino acids' side chains.  Firstly, amino acids do not contain redox-able side chains; their properties would be too reactive.  Also, their side chains are not portable.  So how does it happen?  The answer lies in other enzymatic catalysts.       

Of course, there is always the exception to the rule.  Cysteine is the only amino acid capable of performing redox reactions via the thiol of a cysteine side chain, without the help of enzymatic catalysis (reduction of cysteine shown above). The thiol on the cysteine side chain can be oxidized to a disulfide, and disulfides can be reduced to two thiols. The reduction of the cysteine disulfide can be coupled with oxidation reactions to create a net redox reaction like the one shown below.

Download the chemdraw file above

In this net redox reaction, an alcohol has been oxidized and a disulfide has been reduced. However, this reaction is energetically wasteful. To understand why, one must separately consider the oxidative and reductive half-reactions that have taken place. In the oxidatitve half-reaction, the conversion of a secondary alcohol to a resonance-stabilized oxidative product is concurrent with the release of chemical energy. This oxidative energy release is much greater than the energy required to drive the reductive half-reaction forward; the reduction requires the breaking of the disulfide bond, but electon-electron repulsion between the sulfur lone pairs weakens the disulfide bond (50 kcal/mol) relative to "normal" covalent bonds (90-100kcal/mol). Therefore, due to the signficant difference in redox potentials of each half reaction, the net reaction is characterized by an excess of wasted energy released as heat. 

Cofactors

There are more problems with using amino acid side chains for redox reactions. They lack portability.  Also, if a side chain that was a part of an enzyme took part in a redox reaction, you would kill the enzyme. It is HUGELY energetically wasteful for the body to go through all of the hassle to make this intricate, specific enzyme, only to throw it away over one tiny redox reaction. The name of the game for the body is recycling (Sustainability - it shows up everywhere!). To survive, the body has to be sustainable with the molecules that it works so hard to produce. Therefore using amino acid side chains, and potentially getting valuable enzymes involved, is prohibitively wasteful. Instead, the body needs molecules that it can readily use in many situations to oxidize and reduce substrates. These molecules have to easily reverse into their original state after doing their job. To help solve these problems, evolution has provided more energetically matched reactions with the help of molecules called cofactors. Cofactors are portable, reverse easily, and are "designed" to energetically match certain reactions, preventing energy loss within in a cell. For example, consider the function of NAD+.  

NAD+

NAD+ is an important cofactor because it acts as a portable energy storage.  Specifically, NAD+ is optimally structured to store hydride energy in a portable format, such that NAD+ can gain a hydride ion to become NADH.  This cyclic conversion of NAD+/NADH has been strongly implicated in the component processes of cellular respiration.

Download the chemdraw file above

Examine this diagram and see how the business end of NAD+ can accept just the right amount of energy to lose aromaticity and then rearrange itself to regain the aromaticity and release the energy. Nitrogen is able to withstand a positive charge, and the conjugated pi system of the aromatic ring allows the electrons to flow onto the nitrogen.  If coupled with an secondary alcohol (as in the previously discussed redox reaction), NAD+ reduces to form NADH, while the secondary alcohol is oxidized to form a ketone. Therefore, through the use of a cofactor to mediate organic transformation, nature is able to efficiently catalyze redox reactions.

PLP

Relative to the simple structure of NAD+, pyridoxal phosphate (aka PLP) appears to be a more structurally complex cofactor. However, keep in mind that all 5 PLP functional groups have a unique biological purpose, as determined by billions of years (or 6000, depending on who you ask) of evolution.  PLP is responsible for several reactions that structurally modify amino acids. However, the human body cannot make PLP; it must be biosynthesized after vitamin B6 is ingested via plant products. Thus, since humans are unable to independently synthesize PLP's heterocyclic ring, an innocent salad had to die for your body to efficiently mediate a myriad of organic transformations.

Download the chemdraw file above

PLP-specific enzymes are associated with unique binding pockets on the enzyme's active site that accommodate several of its functional groups. These "docking sites" include (1) an enzymatic salt bridge, used to anchor the negatively charged PLP phosphate site and (2) "hydrophobic" pockets used to anchor the methyl substituent. The electrophillic aldehyde substituent (3) serves as the chemically reactive region of the molecule (i.e. where the fun starts). During the "loading phase" (stay tuned) the aldehyde covalently binds PLP to its enzymatic substrate. The aldehyde's neighboring substituent, a hydroxyl functional group (4), likely forms a hydrogen bond with the reactive aldehyde substituent; this noncovalent interaction serves as a modified form of acid catalysis to speed up reaction rates. The bottom of the aromatic ring (5) is left unbound, acting as "an electron sink" (just means electron density can be dumped there).  Any attempt to structurally stabilize it would hinder lots of interesting chemistry.
Key Points:

• PLP mediates a lot of Redox chemistry because of the reactive aldehyde and the "electron sink."
• Because of noncovalent and covalent interactions with the enzyme's active site, PLP quickly associates with an enzyme and can perform lots of interesting reactions.  A+ for Nature!  Now, let's modify some amino acids.

Mechanism of Action

Loading Phase

The amine from a lysine side chain on the enzyme reacts with the aldehyde (the most reactive part of PLP) and creates an iminium. This allows later reactions to be much faster because an iminium is more reactive (has a higher energy) than an aldehyde. In this reaction a covalent bond is formed because it is more adaptive to bind PLP than to let it be free.

BIG POINT: Don't memorize this mechanism. Rather than pushing arrows, understand the mechanism for what it is (Addition of the enzyme lysine side chain, Proton Transfer, Elimination to the iminium, Proton Transfer, Elimination of the enzyme lysine side chain). The second proton transfer and elimination occur during the preparation phase.

Download the chemdraw file above

This is the iminium.

*Remember to always use reversible arrows on PLP mechanisms*

Preparation Phase

The nitrogen on an amino acid will attack the newly formed iminium. This reaction is a transimination, one nitrogen replaces another. BAM. Now we have a PLP-amino acid adduct.

At this point, you might be on the verge of existential crisis:
 The covalent bond to the enzyme is lost!

But, as Frankie says, RELAX (see 80s dance pop and/or Zoolander for reference). Three additional PLP substituents (the phosphate, methyl, and hydroxyl) are still stuck in the binding pocket of the active site via salt bridging, "hydrophobic" interactions, and either dipole-dipole interactions or hydrogen bonding respecively.(these groups are no longer shown for simplicity, making this depiction of PLP the "business end") . Thus, PLP is locked, loaded, and ready for some seriously cool biochemistry.

Download the chemdraw file above

Now, the PLP Amino Acid Adduct can be modified in four different ways. In each reaction, the last two steps are the:

PLP chemistry or

The turnover phase.

PLP Chemistry

First, there are the non-oxidative products that can be created by the electrons that go down into the "electron sink" of the aromatic ring and then go "all the way back up."

Non-ox. Type 1: A loss of the alpha proton with a non-oxidative product racemizes the amino acid. The R and S products will be produced equally, but often only one is actively used in nature. Note that with a non-oxidative product, the PLP "offloads" by reattaching to the enzyme (this is the turnover phase).

Download the chemdraw file above

Non-ox. Type 2:  Another non-oxidative product is the loss of carbon dioxide. 

Download the chemdraw file above

Non-ox. Type 3:  The last non-oxidative product that can be produced is a side chain loss. This time a proton is removed, however it is not the alpha proton. This still sends electrons down the electron sink and the amino acid is released without its side chain and PLP is re-bound to the enzyme.

Download the chemdraw file above

The next set of reactions are oxidative in nature.

Just like the non-oxidative mechanisms reviewed, oxidative mechanism also include three products: a proton loss, carbon dioxide loss, and a side chain loss. Major Differences from non-oxidative workup:

• In oxidative reactions, the electrons only go "half way up" the sink and the double bond attached directly to the top of the aromatic ring reacts.

• During the turnover phase, instead of being reattached to the enzyme (non-oxidative processes), PLP is converted to PAP via hydrolysis.

Oxidative Type 1: Note that the amino acid is not racemized by the deprotonation of the alpha carbon here like it is in the non-oxidative reaction because a hydrogen is not added back on. This readdition of H does not occur because the electrons only go "half way up."

Download the chemdraw file above

 Oxidative Type 2: Loss of carbon dioxide, electrons go half way up, then hydrolysis seperates the product from PAP.

Download the chemdraw file above

 Oxidative Type 3: Loss of side chain, electrons go half way up, then hydrolysis seperates the product from PAP.

Download the chemdraw file above

 As you can see, the three types of both oxidative and non-oxidative reactions are all very similar. Once you get a little bit of practice which each type these will become very simple.

Pyruvate creation using PLP 

Here is another example of an oxidative PLP mediated reaction that biologically is extremely important:

Download the chemdraw file above

The above reaction is an example of a PLP mechanism that is important in producing pyruvate, an essential molecule in glycolysis.  

Download the chemdraw file above

Download the chemdraw file above

Pyruvate lacks the alpha proton and the side chain of the starting material; for this reason, both of these segments must be removed in the PLP mechanism.  The electrons go half way up before the product release through hydrolysis; therefore, the alpha carbon was oxidized. 

Drill Problems

Answers