Cofactors and Coenzymes

The 20 amino acids that make up proteins offer a surprising range of chemistry: acids, bases, nucleophiles, hydrophobic pockets. But they can’t do everything. They’re poor at carrying electrons one or two at a time, they can’t bind oxygen, and they can’t easily move groups such as methyl or amino groups between molecules. For these jobs, many enzymes recruit a non-protein helper: a cofactor. Understanding cofactors explains why we need trace metals and vitamins, and why deficiencies in them cause such specific diseases.

Vocabulary

• Cofactor: any non-protein component an enzyme needs for activity. Cofactors can be inorganic (metal ions) or organic.
• Coenzyme: an organic cofactor, usually a small molecule, often derived from a vitamin.
• Prosthetic group: a cofactor that is tightly or covalently bound to the enzyme and stays attached throughout the reaction (for example, haem in cytochromes, FAD in some enzymes).
• Cosubstrate: a coenzyme that binds loosely, is changed during the reaction, and then leaves to be regenerated by another enzyme (for example, NAD⁺ and coenzyme A).
• Apoenzyme: the protein part on its own, inactive.
• Holoenzyme: apoenzyme + cofactor, the complete active enzyme.

apoenzyme (inactive) + cofactor → holoenzyme (active)

Metal-ion cofactors

About a third of all enzymes need a metal ion. Metals can do things amino acids can’t:

• Stabilise negative charges, acting as Lewis acids (electron-pair acceptors; see Lewis acids and bases).
• Change oxidation state, carrying electrons in redox reactions (see oxidation and reduction).
• Hold substrates in position through coordination bonds.
• Activate water by lowering its pKa, making a metal-bound hydroxide that’s a powerful nucleophile.

Metal	Example enzyme or protein	Role
Zinc (Zn²⁺)	Carbonic anhydrase, alcohol dehydrogenase, DNA polymerase	Lewis acid; activates water
Magnesium (Mg²⁺)	Hexokinase and most ATP-using enzymes	Binds ATP’s phosphate groups, shielding their charges
Iron (Fe²⁺/Fe³⁺)	Catalase, cytochromes, haemoglobin (not an enzyme)	Redox; binds O₂
Copper (Cu⁺/Cu²⁺)	Cytochrome c oxidase, superoxide dismutase	Redox
Manganese (Mn²⁺)	Arginase; the water-splitting centre of photosynthesis	Redox; Lewis acid
Molybdenum	Nitrogenase, xanthine oxidase	Redox
Selenium (in selenocysteine)	Glutathione peroxidase	Redox
Cobalt (in vitamin B12)	Methylmalonyl-CoA mutase	Rearrangements

Carbonic anhydrase shows a metal at work. Its zinc ion holds a water molecule and makes it much more acidic, so it readily loses a proton to form Zn–OH⁻. That hydroxide attacks carbon dioxide, forming hydrogencarbonate. The enzyme turns over about a million CO₂ molecules per second, and it’s central to how blood carries carbon dioxide and maintains pH (see the blood buffer system).

This is why so many elements are essential in the human body in trace amounts. You can look up the chemistry of each on its element page, for example zinc or molybdenum.

Organic coenzymes and the vitamin connection

Most organic coenzymes are made from water-soluble vitamins, which the body can’t make itself. The vitamin supplies the part of the coenzyme that does the chemistry; the body adds the rest.

Coenzyme	Made from vitamin	What it carries or does	Example process
NAD⁺ / NADH	Niacin (B3)	Hydride ion (2 electrons + H⁺)	Glycolysis, Krebs cycle
NADP⁺ / NADPH	Niacin (B3)	Hydride ion	Fatty acid synthesis, photosynthesis
FAD / FADH₂	Riboflavin (B2)	2 electrons + 2 H⁺	Krebs cycle (succinate dehydrogenase)
Coenzyme A (CoA)	Pantothenic acid (B5)	Acyl groups (e.g. acetyl)	Link reaction, fatty acid breakdown
Thiamine pyrophosphate (TPP)	Thiamine (B1)	Breaks C–C bonds next to C=O	Pyruvate dehydrogenase
Pyridoxal phosphate (PLP)	Pyridoxine (B6)	Amino groups	Amino acid metabolism
Biotin	Biotin (B7)	CO₂	Carboxylation reactions
Tetrahydrofolate	Folic acid (B9)	One-carbon units	DNA base synthesis
Cobalamin coenzymes	B12	Methyl groups; rearrangements	Methionine synthesis

NAD⁺: the electron shuttle

NAD⁺ (nicotinamide adenine dinucleotide) is one of the most important molecules in metabolism. Its nicotinamide ring can accept a hydride ion (H⁻), that is, a proton plus two electrons, from a substrate:

NAD⁺ + H⁻ → NADH

In glycolysis and the Krebs cycle, dehydrogenase enzymes use NAD⁺ to oxidise their substrates. The NADH produced then carries the electrons to the electron transport chain, where their energy is used to make ATP. NAD⁺ is a cosubstrate: it binds, reacts and leaves, and is recycled many times.

Coenzyme A: the acyl carrier

Coenzyme A has a reactive thiol group (–SH) at one end. It forms a thioester bond with acyl groups, such as the two-carbon acetyl group. Thioesters are high-energy bonds, which makes the acetyl group easy to transfer. Acetyl-CoA is the hub of metabolism: carbohydrates, fats and some amino acids are all broken down into acetyl-CoA, which feeds the Krebs cycle or is used to build fatty acids and cholesterol.

PLP: the amino-group handler

Pyridoxal phosphate forms a temporary covalent bond (a Schiff base) with the amino group of an amino acid. This lets enzymes move amino groups between molecules (transamination), remove carboxyl groups, and perform other reactions. PLP-dependent enzymes are involved in making neurotransmitters such as serotonin and GABA.

Deficiency: when cofactors are missing

Because each coenzyme is essential for particular enzymes, a lack of the vitamin produces specific symptoms:

• Thiamine (B1) deficiency causes beriberi, affecting nerves and heart, because pyruvate can’t be fed efficiently into the Krebs cycle. Tissues that depend heavily on glucose, like the nervous system, suffer most.
• Niacin (B3) deficiency causes pellagra: dermatitis, diarrhoea and dementia.
• B12 or folate deficiency causes megaloblastic anaemia, because rapidly dividing cells can’t make DNA properly.
• Iron deficiency causes anaemia and affects iron-containing enzymes.
• Zinc deficiency affects growth, wound healing and immunity.

Vitamin C, though not usually counted as a coenzyme in this sense, is a cofactor for the enzymes that modify proline and lysine in collagen. Without it, collagen is weak and scurvy results.

Cofactors in medicine and industry

• Drug targets: some drugs block cofactor production. Methotrexate (used in cancer and arthritis) blocks the enzyme that regenerates tetrahydrofolate. Some antibiotics block folate synthesis in bacteria.
• Diagnostics: many clinical tests measure NADH, which absorbs ultraviolet light at 340 nm, as a way of following enzyme reactions (see UV–vis spectroscopy).
• Industrial biocatalysis: processes using enzymes that need expensive cofactors like NADH include systems to regenerate the cofactor, so it can be reused thousands of times.

Why evolution uses cofactors

Why don’t enzymes simply build all their chemistry from amino acids? Part of the answer is history. Many coenzymes, including NAD⁺, FAD and coenzyme A, contain pieces that look like RNA nucleotides. This supports the idea that they date from an early stage of life, before proteins took over most catalysis, when RNA molecules may have done the work and used small helper molecules. Proteins that evolved later kept using the same trusted helpers.

The other part of the answer is efficiency. A single coenzyme such as NAD⁺ can serve hundreds of different enzymes, and a single metal ion can give an enzyme chemistry that no amino acid side chain can match. Sharing a small set of versatile cofactors is simpler than evolving a new chemical trick for every enzyme.

Key takeaways

• Cofactors are non-protein helpers; coenzymes are organic cofactors; prosthetic groups stay bound; cosubstrates come and go.
• Metal ions (Zn, Mg, Fe, Cu, Mn, Mo, Se, Co) act as Lewis acids, redox centres and substrate holders.
• Many coenzymes come from B vitamins: NAD⁺ (B3), FAD (B2), CoA (B5), TPP (B1), PLP (B6).
• NAD⁺ carries electrons, CoA carries acyl groups, PLP handles amino groups.
• Missing cofactors cause specific deficiency diseases such as beriberi, pellagra and anaemia. For the enzyme basics, see enzymes: how they work.