6.6: Cofactors and Catalysis - A Little Help From My Friends

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Cofactor Diversity and Chemical Logic

• Distinguish metals from coenzymes as the two categories of enzyme cofactors — identifying common metal cofactors (Fe, Mg, Zn, Cu, Co, Mn, Mo), explaining that coenzymes are small organic molecules usually derived from B-vitamins, classifying coenzymes as loosely bound (dissociable substrates that are regenerated externally) vs. tightly bound prosthetic groups (must be regenerated within the catalytic cycle), and explaining why vitamin deficiencies cause disease by producing inactive apoenzymes — using heme (Fe²⁺ coordinated to a porphyrin ring, bound to histidine residues in succinate dehydrogenase and hemoglobin) as an example of a combined organic/metal cofactor.
• Apply the electron source/sink framework to cofactor-mediated reactions — recognizing that electron sources are typically anions or lone pairs formed after general base deprotonation, that electron sinks include carbonyl oxygens (which accept electrons from breaking C=O π bonds during nucleophilic attack) and, more powerfully, the positively charged nitrogen of an iminium ion — and connect this to the concept that cofactors function by creating superior electron sinks (e.g., converting a carbonyl to an iminium) or by facilitating hydride transfer in anhydrous active sites where free hydride would be impossibly reactive in bulk aqueous solution.

Specific Cofactor Mechanisms

• Explain how thiamine pyrophosphate (TPP, vitamin B₁ derivative) facilitates the decarboxylation of α-keto acids such as pyruvate — describing how the thiazolium ring's C2 carbon (a carbanion stabilized by the adjacent positively charged nitrogen acting as an electron sink) attacks the carbonyl of pyruvate, how CO₂ is released with electrons flowing into the thiazolium ring, and how the resulting hydroxyethyl-TPP intermediate releases acetaldehyde (or is transferred to lipoamide in the pyruvate dehydrogenase complex) — distinguishing this from the spontaneous decarboxylation of β-keto acids, which proceeds without a cofactor because the adjacent ketone already provides an electron sink.
• Explain how FAD and NAD⁺/NADP⁺ mediate oxidation reactions through hydride transfer — distinguishing the tightly bound prosthetic group behavior of FAD (which must be regenerated in situ by a secondary oxidant such as NADP⁺) from the dissociable substrate behavior of NAD⁺/NADP⁺ (which bind and dissociate each catalytic cycle) — and use the oxidation of succinate to fumarate (FAD) and ethanol to acetaldehyde (NAD⁺, catalyzed by alcohol dehydrogenase) as examples, explaining why hydride transfer occurs in the anhydrous enzyme active site rather than in bulk water, and describing the two-step mechanisms for oxidative decarboxylation (hydride transfer creating a carbonyl sink, then decarboxylation) and oxidative deamination (hydride transfer forming a Schiff base, then hydrolysis).
• Explain the exceptional versatility of pyridoxal phosphate (PLP, vitamin B₆ derivative) — describing how PLP's aldehyde reacts with the ε-amino group of an active site Lys to form an aldimine (internal Schiff base) that undergoes transimination with an incoming amino acid substrate (forming an external aldimine), how the protonated iminium nitrogen (pKₐ ~7, so ~50% protonated at physiological pH) serves as a powerful electron sink that activates all three substituents on the α-carbon of the amino acid for reaction — and trace how breaking the bond between the α-carbon and (1) the carboxylate (α-decarboxylation), (2) the β-substituent (β-elimination/serine dehydratase), or (3) the α-hydrogen (racemization or transamination via ketimine formation and tautomerization) gives mechanistically distinct reactions from a single coenzyme platform.

 

Cofactors and Electron Pushing: Sources and Sinks

To make and break bonds, electrons have to be moved. In drawing reaction mechanisms, we showed how electrons move from "sources" to "sinks." In many enzyme-catalyzed reactions, vitamin derivatives are used as substrates or "cofactors" or "coenzymes" to facilitate the flow of electrons in bond-making and breaking. The section focuses on cofactors, which facilitate the flow of electrons from the substrate to the product. We will see these enzymes in more detail in specific chapter sections.

Cofactors are molecules that bind to enzymes and are required for catalytic activity. They can be divided into two major categories: metals and coenzymes. Metal cofactors commonly found in human enzymes include iron, magnesium, manganese, cobalt, copper, zinc, and molybdenum. Coenzymes are small organic molecules that are often derived from vitamins. Coenzymes can bind loosely with the enzyme and be released from the active site. As such, they are also considered reaction substrates. Alternatively, they may be tightly bound and cannot dissociate easily from the enzyme. In this case, after their initial participation in an enzyme-catalyzed reaction, the enzyme would no longer be able to use the cofactor in another round of catalysis until the cofactor returns to its initial state, which requires another chemical reaction and often an additional substrate.

Tight-binding coenzymes are called prosthetic groups. Enzymes not yet associated with a required cofactor are called apoenzymes, whereas enzymes bound with their required cofactors are called holoenzymes. Sometimes, organic molecules and metals combine to form coenzymes, such as in the case of the heme cofactor (Figure 7.15). Coordination of heme cofactors with their enzyme counterparts often involves interactions with histidine residues, as shown in the succinate dehydrogenase enzyme shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): The Heme Cofactor. The family of heme cofactors contains an iron metal coordinated with a porphyrin ring structure, as shown in the left-hand panel within the structure of Heme B. In the right-hand panel, Heme B is shown complexed with the succinate dehydrogenase enzyme from the Kreb Cycle. The structure of Heme B shown in the left-hand panel is from Yikrazuul, and the crystal structure of Succinate Dehydrogenase complexed with Heme B is from Richard Wheeler.

Many biological cofactors are vitamin B derivatives, as shown below in Table \(\PageIndex{1}\). Many vitamin deficiencies cause disease states from inactive apoenzymes that can not function without the correctly bound coenzyme.

Table \(\PageIndex{1}\): Essential B-Vitamins and their Modified Enzyme Cofactors

Cofactors can mediate enzymatic reactions using any of the catalytic strategies listed above. They can serve as nucleophiles, mediate covalent catalysis, form electrostatic interactions with the substrate, and stabilize the transition state. They can also cause strain, distortion, or facilitate acid-base catalysis. Metal-aided catalysis often employs homolytic reaction mechanisms involving radical intermediates. This can be important in reactions, such as those in the electron transport chain, that require the safe movement of single electrons.

We present plausible mechanisms for prototypical reactions using some of the cofactors shown in Table \(\PageIndex{1}\) above. Each shows the flow of electrons from a source to a sink. The source is often a pair of electrons on an anion, formed by the prior removal of a proton from the atom by a general base. A sink could be a carbonyl O, which receives a pair of electrons from one of the C=O bonds of the carbonyl. As a bond is made to the carbonyl, one of the double bonds must break, with the electrons (temporarily, if the reaction is a nucleophilic substitution reaction) going to the carbonyl O, an excellent sink because it is so electronegative. An even better sink is a positive N of an iminium ion; examples are shown below. Just the "business parts" of the cofactors are shown below.

To appreciate the mechanism used by cofactors and show a clear example of an electron source/sink, let's look at a reaction that doesn't require a cofactor, the spontaneous decarboxylation of a β-keto acid, as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\):spontaneous decarboxylation of a β-keto acid

Even though no cofactor is required, nucleophilic catalysis by an amine via Schiff Base formation would accelerate the reaction (as we will see below). Now let's look at how some of the cofactors listed in Table \(\PageIndex{1}\) above facilitate electron flow in reactions.

Thiamine pyrophosphate - decarboxylation of α-keto acids

Thiamine pyrophosphate (TPP) facilitates the decarboxylation of α-keto acids. TPP is a derivative of thiamine (vitamin B1), whose deficiency causes beriberi. TPP is covalently attached to the enzyme, as in pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which catalyze the decarboxylation of α-keto acids. The structure and "business" end of TPP and its catalytic activity are shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Role of TPP in the decarboxylation of pyruvate (step 1) and release of acetaldehyde (step 4)

The number of arrows leading to the product does not reflect the number of steps.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae (1pvd).

Figure \(\PageIndex{3}\): Thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae (1pvd) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...cCq9EJrquwggj8

Flavin Adenine Dinucleotide (FAD) - hydride transfer

FAD and its reduced form, FADH_2, are tightly or covalently attached to an enzyme, so FAD must be regenerated in each catalytic cycle. Figure \(\PageIndex{4}\) shows an example of how this cofactor facilitates the transfer of a :H^- hydride ion to the "business end" of FAD. In contrast to a transfer of protons (H⁺), an acid/base reaction, hydride transfer removes 2 electrons from the substrate (in this case, succinate) along with a proton in an oxidation reaction as FAD is reduced.

Figure \(\PageIndex{4}\): Oxidation of succinate by FAD

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the FAD-binding domain of cytochrome P450 BM3 from Priestia megaterium in complex with NADP⁺ (4DQL)

Figure \(\PageIndex{5}\): FAD binding domain of cytochrome P450 BM3 in complex with NADP⁺ (4DQL). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?hoT1WDCUv1wZFMyRA

FAD is shown in spacefill. NADP⁺, which reoxidizes the reduced FADH₂ back to FAD, is shown in sticks and labeled NAP.

Nicotinamide Adenine Dinucleotide (FAD) reactions

NAD⁺ and a phosphorylated form, NADP⁺, are one of nature's most widely used oxidizing agents and are used as dissociable substrates/cofactors for many different types of enzyme-catalyzed oxidation reactions. The free enzyme is continually active since the NAD⁺ or NADP⁺ cofactor binds (as a substrate) and dissociates (as a product) after each catalytic cycle. The biological synthesis of NAD⁺ requires the vitamin nicotinic acid (niacin), an absence of which causes pellagra.

Oxidation of an alcohol to an aldehyde: The oxidation of ethanol to acetaldehyde by NAD⁺, catalyzed by the enzyme alcohol dehydrogenase, is shown in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Oxidation of ethanol by NAD⁺

The product acetaldehyde contributes to hangovers after ethanol consumption. Note that this reaction is a hydride transfer, which would not be expected to occur in the aqueous environment of a cell, given the extreme reactivity and basicity of a :H^- hydride ion. This transfer occurs in the enzyme's active site, which becomes anhydrous upon binding substrates.

Oxidative decarboxylation of an alcohol: A two-step mechanism for this reaction is shown in Figure \(\PageIndex{7}\)

Figure \(\PageIndex{7}\): Oxidative decarboxylation of an alcohol

After the first step, an electron sink (the carbonyl oxygen) is present at the β-carbon, facilitating the decarboxylation step.

Oxidative deamination of an amine: A two-step reaction, a hydride transfer to form a Schiff base, followed by hydrolysis of the Schiff base, is shown in Figure \(\PageIndex{8}\).

Figure \(\PageIndex{8}\): Oxidative deamination of an amine

We will discuss Schiff base chemistry in more detail below.

Pyridoxal Phosphate Enzymes

Pyridoxal phosphate (PLP) is a derivative of vitamin B6 or pyridoxal. Deficiencies cause convulsions, chronic anemia, and neuropathy. It assists in many reactions (catalyzed by PLP-dependent enzymes). The PLP is bound covalently to lysine residues in a Schiff base linkage (aldimine). This form reacts with many free amino acids (as substrates) to replace the Schiff base to Lys of the enzyme with a Schiff base to the amino acid substrate. First, we will review Schiff base (an imine) formation by the reaction of an aldehyde or ketone with an amine, as shown in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\)

The reaction is essentially a nucleophilic attack by an amine on the carbonyl carbon of an aldehyde or ketone, followed by dehydration. Note that the net effect is to replace one electron sink, a carbonyl (C=O), with an imine (C=NH ↔ C=NH₂⁺), with pKa around 7.0. Hence, 50% of the imine is protonated at neutral pH to form the iminium cation, a much better electron sink than the starting carbonyl!

The structure of pyridoxal phosphate, which contains a reactive aldehyde, is converted to an imine by reaction with the ε-amino side chain of a lysine in the active site of a PLP-dependent enzyme, as shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\)

The figure also shows the replacement of the enzymes' lysine ε-NH₂-PLP bond with a free amino group as an incoming substrate, a process that should proceed with a ΔG⁰ of approximately 0. This occurs in PLP-dependent enzymes with free amino acids as substrates (we will discuss several examples below).

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the E. Coli Aspartate aminotransferase, W140H mutant, maleate complex (1ARI).

Figure \(\PageIndex{11}\): Aspartate aminotransferase, W140H mutant, maleate complex (1ARI). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ZQixXhCwX5EEQ9

Note that the PLP is in Schiff base linkage with the ε-NH₂ group of a lysine in the enzyme's active site.

PLP is quite impressive!

From a chemical perspective, PLP is an ideal molecule for facilitating electron flow in biochemical reactions. William Jencks noted this in his classic text, Catalysis in Chemistry, in which he wrote this elegant description of its properties:

"It has been said that God created an organism especially adapted to help the biologist find an answer to every question about the physiology of living systems; if this is so, it must be concluded that pyridoxal phosphate was created to provide satisfaction and enlightenment to those enzymologists and chemists who enjoy pushing electrons, for no other coenzyme is involved in such a wide variety of reactions, in both enzyme and model systems, which can be reasonably interpreted in terms of the chemical properties of the coenzyme. Most of these reactions are made possible by a common structural feature. That is, electron withdrawal toward the cationic nitrogen atom of the imine and into the electron sink of the pyridoxal ring from the alpha carbon atom of the attached amino acid activates all three of the substituents of this carbon for reactions that require electron withdrawal from this atom."

We'll present three examples of the reaction of an amino acid with a PLP-dependent enzyme. In each case, a different bond to the α-carbon of the amino acid substrate is broken.

α-decarboxylation of an amino acid: Figure \(\PageIndex{12}\) shows a plausible reaction mechanism.

Figure \(\PageIndex{12}\): PLP-dependent decarboxylation of an amino acid.

β-elimination from serine: The enzyme serine dehydratase catalyzes the reaction shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): PLP-dependent β-elimination from serine

Racemization of amino acids: Amino acid racemases use PLP as a cofactor, using a mechanism shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): PLP-dependent racemization of amino acids

Why do racemases exist since the biological world consists of only L-amino acids? There are two possible reasons. Some D-amino acids are found, such as in bacterial cell walls. In addition, amino acids racemize spontaneously, albeit slowly. Reactants with oxygen atoms in the beta-carbon racemize at a much higher rate since they can stabilize the carbanion intermediate formed when the alpha proton is removed during racemization. The concentration of D-Asp and D-Asn can also be used to date biological material. 

Transamination reactions: PLP enzymes also catalyze the transamination reaction, which is shown in Figure \(\PageIndex{15}\)

Amino Acid 1 + α-keto acid 1 ↔ α-keto acid 2 + Amino Acid 2 For example: Figure \(\PageIndex{x}\)

Figure \(\PageIndex{15}\): PLP-dependent transamination reaction

First, Asp, bound to PLP through a Schiff base link, loses the α-H and forms a ketimine through a tautomerization reaction, which ultimately hydrolyzes to form the released oxaloacetate and pyridoxamine. The pyridoxamine reacts with α-ketoglutarate in the reverse of the first three reactions to form Glu.

See Chapter 18.2 for a great description of the role of PLP in transamination reactions.  Here is a link to more mechanistic details of PLP's reactions.

We will explore other cofactors in future chapters.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter extends the analysis of enzyme catalysis mechanisms — general acid/base, nucleophilic, electrostatic, intramolecular catalysis, and transition state stabilization — to include cofactors: small molecules (or metal ions) that bind to enzymes and are essential for catalytic activity because they provide reactive functional groups or electronic properties that amino acid side chains alone cannot supply.

Cofactor organization divides into two broad categories. Metal cofactors (Fe, Zn, Mg, Cu, Co, Mn, Mo) participate in Lewis acid catalysis, stabilize charged intermediates and transition states, mediate homolytic (radical) reactions in electron transport, and maintain active site geometry. Organic coenzymes are mostly derivatives of B-vitamins; vitamin deficiencies therefore produce disease by rendering specific enzymes inactive in their apoenzyme form (without cofactor) rather than their active holoenzyme form (with cofactor). Coenzymes that dissociate after each catalytic cycle act as substrates or cosubstrates (NAD⁺, NADP⁺, SAM, CoA); those that remain bound between cycles are prosthetic groups and must be regenerated within the catalytic cycle (FAD, PLP, TPP). Heme — an iron ion coordinated to protoporphyrin IX, typically bound to enzymes through histidine coordination — exemplifies how metals and organic scaffolds combine to create versatile cofactors capable of reversible redox chemistry (hemoglobin, cytochrome c) and catalysis (peroxidases, cytochrome P450s).

The electron source/sink framework unifies the mechanistic understanding of cofactor-assisted reactions. Electrons flow from nucleophilic sources (lone pairs on anions generated by deprotonation, or π-bond electrons on enolates) toward electrophilic sinks. Carbonyl oxygens are good sinks because they can transiently accept electrons as nucleophiles attack the adjacent carbon. Iminium ions (protonated Schiff bases, C=NH₂⁺) are superior sinks because the full positive charge on nitrogen provides even greater electrophilic activation of the adjacent carbon. Most cofactor-dependent mechanisms involve creating or using such enhanced electron sinks to facilitate reactions — decarboxylation, transamination, β-elimination, racemization, oxidation — that would otherwise be too slow or require prohibitively reactive intermediates.

Thiamine pyrophosphate (TPP), the active form of vitamin B₁ (beriberi disease on deficiency), facilitates the decarboxylation of α-keto acids such as pyruvate. The key feature is the thiazolium ring's C2 carbon, which is thermodynamically prone to forming a carbanion stabilized by the adjacent positively charged nitrogen — itself an excellent electron sink. The C2 carbanion attacks the α-keto carbonyl of pyruvate; electron flow through the developing negative on the ketone oxygen facilitates C–C bond cleavage and CO₂ loss, generating a hydroxyethyl-TPP intermediate that then releases acetaldehyde (or proceeds to acyl transfer in the pyruvate dehydrogenase complex). This mechanism differs fundamentally from the spontaneous β-keto acid decarboxylation (which requires no cofactor because the β-keto group already provides the electron sink) — TPP creates an equivalent electron sink adjacent to an α-keto acid, a substrate that otherwise lacks the correct arrangement for facile decarboxylation.

FAD is a tightly bound prosthetic group that accepts a hydride ion (:H⁻) plus effectively removes two electrons from a substrate (net oxidation) as it is reduced to FADH₂. The oxidation of succinate to fumarate (in the citric acid cycle, catalyzed by succinate dehydrogenase) illustrates this: the C–H bond on C2 of succinate is broken, hydride is transferred to the N5 of the isoalloxazine ring of FAD, and the double bond of fumarate is generated. Since FADH₂ cannot dissociate and be replaced by fresh FAD, the enzyme requires a coupled oxidation step (typically by NAD⁺ or in the electron transport chain) to regenerate FAD before another catalytic cycle. NAD⁺/NADP⁺ (derived from niacin, whose deficiency causes pellagra) are dissociable substrates that accept hydride at the C4 of the nicotinamide ring in a stereospecific fashion. Alcohol dehydrogenase exemplifies this: NAD⁺ accepts a hydride from the Zn²⁺-activated α-carbon of ethanol in the anhydrous active site, generating acetaldehyde and NADH. The anhydrous environment is essential because hydride transfer in bulk water would be thermodynamically unfavorable. Two additional mechanistic patterns emerge: oxidative decarboxylation (first oxidation by NAD⁺ creates a carbonyl electron sink adjacent to the carboxylate, which then spontaneously decarboxylates) and oxidative deamination (hydride transfer generates an iminium/Schiff base intermediate that is subsequently hydrolyzed to release the keto acid and NH₃).

Pyridoxal phosphate (PLP), the active form of vitamin B₆ (deficiency causes convulsions, anemia, and neuropathy), is one of the most versatile coenzymes known, participating in transamination, decarboxylation, β-elimination, and racemization of amino acids — all from a single chemical platform. PLP's aldehyde reacts with the ε-amino group of an active site Lys to form an internal Schiff base (aldimine). When an amino acid substrate arrives, it undergoes transimination — displacing the Lys ε-amino group with the substrate's α-amino group to form an external aldimine with ΔG° ≈ 0. The protonated iminium nitrogen of this external aldimine (pKₐ ~7, so approximately half-protonated at physiological pH) is a powerful electron sink that activates all three substituents at the α-carbon. Which bond to the α-carbon is broken determines the reaction: cleavage of the α-C–carboxylate bond gives α-decarboxylation (producing biogenic amines like dopamine and GABA); cleavage of the α-C–β-substituent bond gives β-elimination (serine dehydratase converting serine to pyruvate + NH₃); and cleavage of the α-C–H bond gives either racemization (reprotonation from the opposite face in amino acid racemases, important for D-amino acids in bacterial cell walls) or transamination (tautomerization through the ketimine intermediate and hydrolysis to release the α-keto acid plus pyridoxamine, which then aminates a second α-keto acid in the reverse reaction). William Jencks captured the chemical elegance of PLP: no other coenzyme participates in so wide a range of reactions, all interpretable through the single unifying principle of electron withdrawal from the α-carbon toward the iminium nitrogen and pyridoxal ring electron sink.