6.6. Cofactors
Many enzymes work using only the functional groups on the side chains of the amino acids that they are made of. For example, serine proteases (see Section 6.5.3) use the side chains of their amino acids to perform their function. However, some enzymes require other compounds in order to catalyze their reactions. For example, zinc metallo-proteases (see Section 6.5.5) need the zinc cation in order to function. Without the zinc cation it cannot break the amide bonds.
A cofactor is a compound other than the substrates themselves that is required for an enzyme to catalyze its reaction. There are two broad categories that all cofactors can be grouped into (see Sections 6.6.1 and 6.6.2). Technically, there are more specific subdivisions depending on the exact function and structure of the cofactor. These are not required at an introductory level. However, they may be encountered in other sources.
In some cases the cofactor needed is simply a metal ion. These are commonly referred to as “essential ions”. Essential ions typically function in straightforward ways, most commonly as Lewis acids/bases for catalysis by metal ion interactions. There are exceptions where the essential ion also participates in chemical reactions. Typically these involve the reduction/oxidation of compounds (REDOX reactions).
In some cases the cofactor needed is an organic compound. These are commonly referred to as “coenzymes”. Unlike essential ions coenzymes have a wide array of structures and functions. They may be used in straightforward or exceptionally complex ways. Many vitamins are, or are used to make, coenzymes.
Because of the high variability in coenzyme structure and function a generalized discussion is not typically useful. Instead, a set of examples are provided to highlight the more common (and straightforward) coenzymes.
6.6.2.1. Example: Thiamine Pyrophosphate (TPP)
One of the common coenzymes is thiamine pyrophosphate (sometimes abbreviated as TPP). Its structure consists of a diphosphate (referred to by biochemists as pyrophosphate) attached to a thiazolium ring, which is also attached to a pyrimidine ring (Figure 6.13). It is made from Vitamin B1.
Figure 6.13 – Structure of Coenzyme Thiamine Pyrophosphate (TPP).
The CH of the thiazolium ring is slightly acidic. When a base removes the proton it forms an unusual zwitterionic structure (Scheme 6.15). Some sources refer to this as an “ylide”: a formally negatively charged atom (usually a carbanion) directly attached to a heteroatom with a formal positive charge (usually nitrogen, sometimes phosphorus or sulfur) where both atoms have a full octet. Other sources use the term “carbene” (the specific type of ylide this has). Ylides have unusual chemical reactivities. The ylide’s special reactivity is what thiamine pyrophosphate is used for as a coenzyme.
Scheme 6.15 – Generalized Deprotonation of Thiamine Pyrophosphate to form an Ylide/Carbene.
6.6.2.1.1. TPP and Decarboxylation of Pyruvic Acid
Thiamine pyrophosphate has several common and/or important uses. One of these is as part of general cell metabolism: the decarboxylation of pyruvic acid (2-oxopropanoic acid) in pyruvate decarboxylase enzymes. Pyruvic acid is a small but important compound produced and used in several metabolic pathways. The enzyme uses TPP to perform the reaction (Scheme 6.16). The enzyme still has the standard binding-reaction-release stages. Typically it also has other interactions, such as hydrogen bonding with side chains and/or NH’s, helping the reaction. For clarity these are omitted from the reaction mechanism.
Scheme 6.16 – Example of a Simplified Catalytic Cycle/Mechanism for Pyruvate Decarboxylase with Thiamine Pyrophosphate Coenzyme.
6.6.2.1.2. TPP and Transketolases
Thiamine pyrophosphate has several common and/or important uses. One of these is modifying/interconverting monosaccharides: chain shortening and lengthening in transketolase enzymes. This takes two carbons from a ketose (making a shorter aldose) and attaches them to an aldose (making a longer ketose). The enzyme uses TPP to perform the reaction (Scheme 6.17). The enzyme still has the standard binding-reaction-release stages. Typically it also has other interactions, such as hydrogen bonding with side chains and/or NH’s, helping the reaction. For clarity these are omitted from the reaction mechanism.
Scheme 6.17 – Example of a Simplified Catalytic Cycle/Mechanism for Transketolase with Thiamine Pyrophosphate Coenzyme.
6.6.2.1.3. TPP and Benzoin Reactions
Thiamine pyrophosphate has several common and/or important uses. One of these is to combine two aldehydes and form alpha-hydroxy ketones (α-hydroxy ketones). Some sources refer to this as the “benzoin reaction”. These products have various biological functions but are generally challenging to synthesize without using ylides. Several different types of enzymes perform this reaction depending on the exact aldehydes being combined. The enzymes use TPP to perform the reaction (Scheme 6.18). The enzymes still have the standard binding-reaction-release stages. Typically they also have other interactions, such as hydrogen bonding with side chains and/or NH’s, helping the reaction. For clarity these are omitted from the reaction mechanism.
Scheme 6.18 – Example of a Simplified Catalytic Cycle/Mechanism for the Benzoin Reaction with Thiamine Pyrophosphate Coenzyme.
6.6.2.2. Example: Tetrahydrofolic acid (THFA)
One of the common coenzymes is tetrahydrofolic acid (sometimes abbreviated as THFA). Its structure consists of a pterin heterocycle (the trivial name for this specific heterocyclic ring system) attached to para-amino benzoic acid, which is also attached to glutamic acid through an amide bond (Figure 6.14). It is made from Vitamin B9. The “tetrahydro-” prefix relates it to the structure of the vitamin.
Figure 6.14 – Structure of Coenzyme Tetrahydrofolic Acid (THFA).
6.6.2.2.1. THFA and Methyl/Methene/Formyl Transfers
Two of the NH’s of tetrahydrofolic acid have unusual reactivity. They are able to be functionalized to have various carbon-containing groups added to them (Figure 6.15). Exactly how this occurs varies by the specific group and position. Most reactions that generate these compounds are complex and/or require several enzymes and multiple steps.
Figure 6.15 – Examples of Functionalized Tetrahydrofolic Acid (THFA) Derivatives used by a Variety of Enzymes.
Once the nitrogen(s) have been functionalized they are able to add the carbon-containing group to a different molecule, often an amino acid or a nucleic acid. Again, the specifics of how these reactions occur are typically complex and/or require several enzymes and multiple steps, particularly to regenerate the catalyst. However, the ability to gain and transfer these carbon-containing groups is what tetrahydrofolic acid is used for as a coenzyme. For example, the synthesis of thymine from uracil (important components of DNA and RNA respectively) proceeds through a tetrahydrofolic acid-mediated reaction (Scheme 6.19).
Scheme 6.19 – Example of a Simplified Reaction Diagram for Conversion of Uracil to Thymine with Tetrahydrofolic Acid Coenzyme.
6.6.2.3. Example: S-Adenosyl Methionine (SAM)
One of the common coenzymes is S-adenosyl methionine (sometimes abbreviated as SAM). Its structure consists of a methionine attached to a ribofuranose ring, which is also attached to an adenine ring (Figure 6.16). The combination of a ribofuranose and adenine is sometimes referred to collectively as an “adenosine” instead. It is made from adenosine triphosphate (ATP).
Figure 6.16 – Structure of S-Adenosyl Methionine (SAM).
6.6.2.3.1. SAM-Dependent Methylases
The methyl group on the sulfur of S-adenosyl methionine is highly reactive. SAM is able to add a methyl group to various compounds as part of so-called SAM-dependent methyltransferase enzymes. Many such enzymes exist, often with extremely specific substrate selectivity; typically each of these enzymes can add a methyl group to only a single type of molecule. Unlike with tetrahydrofolic acid (see Section 6.6.2.2.1), the specifics of how these reactions occur are typically simple: the methyl (electrophile) is attacked by a nucleophile and becomes part of the product. However, like THFA regenerating S-adenosyl methionine is complex and involves several enzymes and multiple steps. The ability add a methyl group to other compounds is what S-adenosyl methionine is used for as a coenzyme. For example, the synthesis of epinephrine (also known as adrenaline) from norepinephrine proceeds through a S-adenosyl methionine-dependent methyltransferase reaction (Scheme 6.20).
Scheme 6.20 – Example of a Simplified Reaction Diagram for Conversion of Norepinephrine to Epinephrine (Adrenaline) with S-Adenosyl Methionine Coenzyme.
6.6.2.4. Example: Nicotinamide Adenine Dinucleotide (NAD+/NADH)
One of the common coenzymes is nicotinamide adenine dinucleotide (sometimes abbreviated as NAD+ or NADH depending on the form). Its structure consists of an adenine ring attached to a ribofuranose ring, which is also attached to a diphosphate, which is also attached to a ribofuranose ring, which is finally attached to nicotinamide (Figure 6.17). The combination of a nitrogen-containing heterocycle, a ribofuranose, and a phosphate is sometimes referred to collectively as a “nucleotide”, leading to the “dinucleotide” portion of the name; the molecule is the combination of two nucleotides, one from nicotinamide and one from adenine. It is made from Vitamin B3.
Figure 6.17 – Structure of Coenzyme Nicotinamide Adenine Dinucleotide (NAD+).
The nicotinamide portion has a rare type of reactivity. When it is cationic (NAD+) it can accept a hydride from another molecule (Scheme 6.21). This removes the aromaticity but also negates the cationic charge. Conversely, the neutral form (NADH) can donate a hydride to another molecule. This is typically phrased in terms of REDOX reactions: the cationic form (NAD+) is an oxidant (accepts electrons from something that loses electrons); the neutral form (NADH) is a reductant (donates electrons to something that gains electrons). The nicotinamide’s special reactivity is what nicotinamide adenine dinucleotide is used for as a coenzyme.
Scheme 6.21 – Generalized Interconversion of Cationic NAD+ (Oxidant) and Neutral NADH (Reductant) Forms of Nicotinamide Adenine Dinucleotide.
6.6.2.4.1. NAD+/NADH and Dehydrogenation/Hydrogenation of Pyruvate (2-Oxopropanoic Acid)
The interconversion of NAD+ and NADH is fully reversible and (usually) both reactions are taken advantage of. For example, the synthesis of pyruvic acid (2-oxopropanoic acid) from lactic acid ((S)-2-hydroxypropanoic acid) proceeds through a NAD+/NADH-mediated reaction with the enzyme lactate dehydrogenase (Scheme 6.22). The same enzyme also catalyzes the reverse process depending on which compound the cell requires (or has excess of).
Scheme 6.22 – Example of a Simplified Reaction Diagram for Interconversion of Lactic and Pyruvic Acid with Nicotinamide Adenine Dinucleotide Coenzyme.
6.6.2.5. Example: Flavin Adenine Dinucleotide (FAD/FADH2)
One of the common coenzymes is flavin adenine dinucleotide (sometimes abbreviated as FAD or FADH2 depending on the form). Its structure consists of an adenine ring attached to a ribofuranose ring, which is also attached to a diphosphate, which is also attached to ribitol (reduced ribose), which is finally attached to flavin (Figure 6.18). As with NAD+/NADH the molecule is the combination of two nucleotides, one from flavin and one from adenine. It is made from Vitamin B2.
Figure 6.18 – Structure of Coenzyme Flavin Adenine Dinucleotide (FAD).
The flavin portion has multiple rare types of reactivity. The FAD form can accept two electrons and two hydrogens, forming the FADH2 form. The reverse is also possible. This is similar to nicotinamide adenine dinucleotide but potentially more complex: these are often single-electron-transfer reactions (sometimes abbreviated as SET reactions; also known as radical reactions) NOT hydride accepting/donating (Scheme 6.23). Using single-electron-transfer reactions makes FAD/FADH2 much more reactive than NAD+/NADH but also makes most mechanisms with this type of reactivity significantly more complex. Radical reactions (and their mechanisms) fall outside the scope of this text and are normally discussed in advanced courses.
Scheme 6.23 – Generalized Interconversion of FAD and FADH2 Forms of Flavin Adenine Dinucleotide.
Alternatively, the FAD form can accept two electrons and two hydrogens, forming the FADH2 form, in other indirect ways (see Section 6.6.2.5.1). These are again not typically hydride accepting/donating, but may involve nucleophile-electrophile and/or acid-base reactions. The multiple complex ways of accepting two electrons and two hydrogens is what flavin adenine dinucleotide is used for as a coenzyme.
6.6.2.5.1. FAD/FADH2 and Amino Acid Oxidases
The interconversion of FAD and FADH2 is fully reversible and (usually) both reactions are taken advantage of. However, often only one of the two paths is commonly used and other ways of regenerating the catalyst are required. For example, the metabolism of certain excess amino acids proceeds through a FAD/FADH2-mediated reaction with enzymes called amino acid oxidases (Scheme 6.24). This converts the amine group to a ketone. For example, excess alanine ((S)-2-aminopropanoic acid) is oxidized to pyruvic acid (2-oxopropanoic acid), which may then be used to make other compounds.
Scheme 6.24 – Example of a Simplified Reaction Diagram for Digestion of Alanine to Pyruvic Acid with Flavin Adenine Dinucleotide Coenzyme.
The same enzymes also catalyze the reverse process depending on which compound the cell requires (or has excess of). However, typically it digests the amino acids and requires a different compound be reduced to regenerate the FAD coenzyme. Often this is oxygen. Because the overall reaction takes three common compounds (an amino acid, water, and oxygen) and produces three potentially toxic compounds (an acid, ammonia, a peroxide) this enzyme has another use: it is present in large quantities in certain kinds of snake venom.
6.6.2.6. Example: Coenzyme A (CoA)
One of the common coenzymes is simply referred to as Coenzyme A (sometimes abbreviated as CoA, rarely abbreviated as HSCoA). Its structure consists of an adenine ring attached to a phosphorylated ribofuranose ring, which is also attached to a diphosphate, which is finally attached to a pair of small biomolecules (pantothenic acid and cysteamine; Figure 6.19). It is made from Vitamin B5.
Figure 6.19 – Structure of Coenzyme A (CoA).
Coenzyme A is both highly versatile and widely used in many metabolic pathways. CoA is synthesized and used by every organism whose genome has been studied. The SH of the cysteamine can be functionalized several different ways, typically becoming a thioester (Figure 6.20). The exact way these thioesters are formed varies considerably; there are many different possible sources of the new atoms and many different ways they can be added to CoA’s sulfur. These thioesters are then used to transfer their carbonyl-containing groups to other molecules (e.g. convert an amine to an amide, convert an alcohol to an ester, etc.). Because sulfur is a much better leaving group than oxygen or nitrogen the transfer of groups becomes easier. The ability to carry and transfer carbonyl-containing groups to other molecules is what Coenzyme A is used for as a coenzyme.
Figure 6.20 – Generalized Functionalized Coenzyme A (R-CoA).
6.6.2.6.1. CoA and Fatty Acid Synthesis
Coenzyme A has several common and/or important uses. One of these is as part of general cell metabolism: all fatty acid synthesis proceeds through Coenzyme A-mediated processes. These paths vary considerably depending on the organism and the exact fatty acid being synthesized. They typically involve a large number of different enzymes performing very specific roles and often require multiple other cofactors during the process. As a general example (Scheme 6.25), two functionalized CoA coenzymes are combined to begin the synthesis. This proceeds through an aldol-type reaction. Modification (typically removal of the carbonyl) and addition of more carbons to the chain then occurs, before the fatty acid is finally released. The overall process involves multiple CoA cofactors, multiple other cofactors, and over a dozen enzymes.
Scheme 6.25 – Example of a Simplified Process for Fatty Acid Synthesis with Coenzyme A.
6.6.2.6.2. CoA and the Citric Acid Cycle
Coenzyme A has several common and/or important uses. One of these is as part of general cell metabolism: the citric acid cycle (sometimes called the Krebs cycle) proceeds through Coenzyme A-mediated processes. The citric acid cycle is the general process by which all oxygen-breathing organisms convert fats, sugars, and/or amino acids into energy for the cell. It is also one of the ways by which those cells synthesize other compounds, such as certain amino acids. The process relies on Coenzyme A as well as several other cofactors (Figure 6.21). The cycle itself is lengthy, complex, and involves many enzymes and cofactors. In reality, the cycle is further complicated by variability: different organisms may use different enzymes (and slightly different mechanisms) for certain steps, and often there are multiple possible ways for any given step to occur within a single cell. Understanding the citric acid cycle lies outside the scope of this text. This image is provided only to illustrate the importance of CoA to the process.
Figure 6.21 – Stylized Representation of the Citric Acid Cycle.
6.6.2.6.3. CoA and Acetyltransferases
Coenzyme A has several common and/or important uses. General acetylation proceeds through Coenzyme A-mediated processes. Several different enzymes catalyze these reactions depending on exactly which compound(s) they are modifying, but they are generally referred to as acetyltransferases. The interconversion is typically reversible and (usually) both reactions are taken advantage of. For example, the synthesis of acetylcholine from choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) proceeds through a Coenzyme A-mediated reaction with the enzyme “choline acetyltransferase” (Scheme 6.26). The same enzyme also catalyzes the reverse process depending on which compound the cell requires (or has excess of). Choline has several general uses (e.g. in cell membranes) and acetylcholine is a neurotransmitter (part of the autonomous nervous system, muscle movement, “mood”). Because both of these molecules have important but very different roles the cell uses CoA to rapidly interconvert them as needed.
Scheme 6.26 – Example of a Simplified Reaction Diagram for Interconversion of Choline and Acetylcholine with Coenzyme A.
6.6.2.7. Example: Pyridoxal Phosphate (PLP)
One of the common coenzymes is pyridoxal phosphate (sometimes abbreviated as PLP). Its structure consists of a phosphate attached to a pyridoxal ring (Figure 6.22). It is made from Vitamin B6.
Figure 6.22 – Structure of Coenzyme Pyridoxal Phosphate (PLP).
The alcohol of the pyridoxal ring is slightly acidic, while the nitrogen is basic. As a result, PLP often exists as a zwitterionic structure (Scheme 6.27). While in the zwitterionic form the pyridoxal ring has special properties and can accept electrons from groups attached to the aldehyde. This special reactivity is what pyridoxal phosphate is used for as a coenzyme.
Scheme 6.27 – Equilibrium Between Two Forms of Pyridoxal Phosphate.
6.6.2.7.1. PLP and Amino Acid Decarboxylases
Pyridoxal phosphate has several common and/or important uses. One of these is metabolizing amino acids: decarboxylation in amino acid decarboxylase enzymes. This removes the carboxylic acid groups from the amino acid, forming a new compound that may be used by the cell for other purposes. The enzyme uses PLP to perform the reaction (Scheme 6.28). The enzyme still has the standard binding-reaction-release stages. Typically it also has other interactions, such as hydrogen bonding with side chains and/or NH’s, helping the reaction. For clarity these are omitted from the reaction mechanism.
Scheme 6.28 – Example of a Simplified Catalytic Cycle/Mechanism for an Amino Acid Decarboxylase with Pyridoxal Phosphate Coenzyme.
The entire process is fully reversible; the same enzyme may be used to synthesize an amino acid by performing the same steps in reverse.
6.6.2.7.2. PLP and Transaminases
Pyridoxal phosphate has several common and/or important uses. One of these is synthesizing/metabolizing amino acids: transferring an amine from one amino acid to a new one in transaminase enzymes. This takes a pre-existing amino acid and converts it to an alpha-keto acid (α-keto acid), and then takes a different alpha-keto acid and converts it into a new amino acid. The enzyme uses PLP to perform the reaction (Scheme 6.29). The enzyme still has the standard binding-reaction-release stages. Typically it also has other interactions, such as hydrogen bonding with side chains and/or NH’s, helping the reaction. For clarity these are omitted from the reaction mechanism. The overall reaction mechanism is exceptionally long but not conceptually difficult: the second half of the cycle is just the reverse of the first half (the opposite steps in the opposite order).
Scheme 6.29 – Example of a Simplified Catalytic Cycle/Mechanism for an Amino Acid Transaminase with Pyridoxal Phosphate Coenzyme.
