18.4: An Overview of Amino Acid Metabolism and the Role of Cofactors

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Learning Goals 

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

Amino Acid Carbon Skeleton Catabolism

• Classify all 20 amino acids as glucogenic, ketogenic, or both, using structural reasoning — including chain length, oxidation state, and the nature of the carbon skeleton — to predict whether an amino acid's catabolism produces pyruvate, TCA intermediates (α-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate), acetyl-CoA, or acetoacetyl-CoA, and explain why only acetyl-CoA-producing amino acids are purely ketogenic.
• Explain the concept of anaplerotic reactions in the context of amino acid catabolism, describing how glucogenic amino acids that enter the TCA cycle as intermediates replenish the cycle and can support net glucose synthesis through gluconeogenesis, while purely ketogenic amino acids cannot.

One-Carbon Metabolism: Tetrahydrofolate and SAM

• Describe the structure of tetrahydrofolate (FH₄) as a carrier of one-carbon units in three oxidation states (+2 as formyl/methenyl, 0 as methylene, and −2 as methyl), explain how these derivatives are interconverted using NAD(P)H-dependent oxidoreductions, and identify the biosynthetic products — thymidine, purines, serine, and methionine — that each 1C-FH₄ derivative contributes to.
• •Explain the dual-cofactor mechanism of serine hydroxymethyltransferase (SHMT), describing how PLP facilitates α-elimination of the serine side chain and how FH₄ captures the resulting formaldehyde as N⁵,N¹⁰-methylene-FH₄, and explain why SHMT is a target for malaria treatment.
• Describe how dihydrofolate reductase (DHFR) sequentially reduces folate to dihydrofolate and then to tetrahydrofolate using two NADPH molecules, explain why this enzyme is a target for cancer chemotherapy, and describe how methotrexate's structural similarity to folate enables competitive inhibition.
  • Explain the methionine cycle, tracing the path from N⁵-methyl-FH₄ → methylcobalamin → methionine → SAM → S-adenosylhomocysteine → homocysteine → methionine, explaining why SAM is a more potent methyl donor than N⁵-methyl-FH₄ (using the S_N2 mechanism and the sulfonium electron sink), and why vitamin B12 deficiency leads to accumulation of N⁵-methyl-FH₄, depletion of other FH₄ derivatives, and megaloblastic anemia.
• Explain how methylenetetrahydrofolate reductase (MTHFR) is allosterically inhibited by SAM as part of a feedback loop that prevents depletion of the folate 1C pool when methylating capacity is already sufficient, and describe the clinical significance of the serine-glycine one-carbon (SGOC) cycle in tumor cell proliferation.

Amino acid degradation

In the previous chapter, we saw how nitrogen is removed from amino acids to produce urea or NH₄⁺. What are the fates of the remaining carbon skeletons? This section is where students might get overwhelmed by the diversity of amino acid degradation pathways, so it helps to realize that the carbon skeletons of deaminated amino acids can be used for biosynthesis or energy production and are converted to key glycolytic and TCA intermediates that you have seen many times before. Everything is interconnected, which makes the study of metabolism both daunting and fascinating, as organisms strive to extract all the energy and molecule-building atoms from a metabolite while minimizing waste.

Here are some key features of amino acid catabolism:

• Some are converted to pyruvate, the end product of glycolysis and the starting reactant of gluconeogenesis. Hence, these amino acids are glucogenic.
• Some are converted to acetoacetyl-CoA or acetyl-CoA. Both can be converted to ketone bodies (acetoacetate and β-hydroxybutyrate), which are considered ketogenic. Since the two carbons of the acetyl group of acetyl-CoA are lost as CO₂ in the TCA cycle, and there is no reverse for the pyruvate dehydrogenase reaction (pyr → acetyl-CoA), acetyl-CoA formed by amino acid degradation can not be used to create glucose in a net fashion.
• Some are metabolized to form TCA intermediates. Since they are added in a net fashion to the TCA cycle and don't remove the existing pool of TCA intermediates, they can produce, in a net fashion, directly or indirectly, molecules that can be used to produce glucose. These entry reactions into the TCA cycle, which replenish or add to TCA intermediates, are called anaplerotic (replenishing) reactions. Hence, these amino acids are also glucogenic.
• Some have multiple degradation pathways and can produce both acetyl-CoA and pyruvate, so they are both glucogenic and ketogenic.

Let's get more explicit:

• purely ketogenic: only Leu and Lys (the only amino acids whose names start with L, and you have to Love them since there are only two amino acids in this category)
• both: 5, including the aromatics - Trp, Tyr, Phe - and Ile/Thr
• purely glucogenic: the rest

General hints if an amino acid is glucogenic and/or ketogenic are derived from its structure.

Fully or Partly Ketogenic Amino Acids: Amino acids, except for Arg, that have a continuous chain of 3 or more carbon atoms in their side chains and hence are more "fat-like" are ketogenic or ketogenic/glucogenic. These include Lys, Leu, Ile, and the aromatic amino acids Phe, Trp, and Tyr, excluding Val. (Note: Thr, both ketogenic and glucogenic, doesn't fit this rule). The wholly or partly ketogenic amino acids are shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Fully or Partly Ketogenic Amino Acids

Purely Glucogenic Amino Acids (for Pyr and/or TCA intermediates): Which amino acids produce which intermediate? Except for glycine, it's pretty easy to remember. The number of carbons in the intermediate formed is the same as the number of carbons in the longest chain in the amino acid.

These amino acids form the 5C TCA intermediate alpha-ketoglutarate (2-oxoglutarate) and are shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Glucogenic Amino Acids Converted to 5C α-ketoglutarate (2-oxoglutarate)

These amino acids form either the 4C TCA intermediate succinate or oxaloacetate. Amino acids with more oxidized 4C atoms in the continuous chain produced oxaloacetate (which is more oxidized than succinate), while the least oxidized ones produced succinic acid (which is more reduced than oxaloacetate). These amino acids are shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Glucogenic Amino Acids Converted to 4C oxaloacetate and succinate

The 3C amino acids, Ser, Ala, and Cys, form pyruvate, as shown in Figure \(\PageIndex{4}\).  The only 2C amino acid, glycine, does as well.

Figure \(\PageIndex{4}\): Glucogenic Amino Acids Converted to 3C pyruvate

A full figure summarizing everything above is shown in Figure \(\PageIndex{5}\)!

Figure \(\PageIndex{5}\): Summary of amino acid breakdown products

With a little help from my friends: Cofactors and Amino Acid Catabolism

The myriad of breaking, making, and rearranging carbon atoms in amino acid catabolism is daunting. As with all reactions, a pathway for the flow of electrons and for the stabilization of transition states and intermediates must be in place for the reactions to occur. All methods of catalysis (general acid/base, covalent/nucleophilic, electrostatic/metal ion, and preferential stabilization of the transition state) occur. Yet some reactions need additional "helpers" or "cofactors" to facilitate electron flow and shuttle small motifs (from electrons in redox reactions to methyl groups in methylases) from one molecule to another. In the previous section, we saw the important role of pyridoxal phosphate in transaminases and aminotransferases. Even the terminology cofactor is a bit confusing since analogous terms are used in different contexts:

• cofactors - nonprotein small molecules or ions (divalent ions, transition metal ions, and Fe/S clusters, for example) that must bind to an enzyme but usually stay put once bound. That is, they don't dissociate during the catalytic cycle. For instance, PLP must initially bind to an enzyme, then becomes covalently attached via a Schiff base to the epsilon amino group of a Lys in the catalytic site. For example, that linkage might be swapped with an incoming amino acid substrate but reform in the catalytic cycle, allowing the enzyme to remain functional.
• coenzyme - a vitamin derivative, organic cofactor, as compared to inorganic ions. The term "coenzyme" is a poor historical name that persists.
• prosthetic group - this is usually a coenzyme that is either covalently bound (like PLP) or noncovalently bound with such a low K_D (like FAD/FADH₂-containing enzymes) that they stay bound
• cosubstrate - these bind as substrate (for example, NAD⁺), and depart as products (for example, NADH).

Here is a table of common nonmetallic cofactors.

Cofactor	Vitamin Derivative	Carrier
biotin	H - biotin	1 C - CO2 (most oxidized)
tetrahydrofolic acid (FH4 or THF)	B9 - folic acid	1C - formyl, -(C=O)H, methylene (-CH2) (more reduced) and methyl -CH3
cobalamin	B12 - cobalamin	methyl CH3
thiamine pyrophosphate	B1 - Thiamine	2C group
coenzyme A	B5 - pantothenic acids	acetyl (CH3-C=O) and acyl (R-C=O)
pyridoxal phosphate	B6 - pyridoxine	amino and carboxyl groups
NAD^+/NADP^+	B3 - Niacin	electrons
FAD/FMN	B2 - riboflavin	electrons

Other non-vitamin cofactors include S-adenosylmethionine (SAM or adoMet), a carrier of methyl groups; Coenzyme Q, a carrier of electrons; tetrahydrobiopterin, a carrier of oxygen and electrons; and heme, a carrier of electrons.

We've already described amino acid deamination reactions using PLP. Now let's consider the biochemistry of two of these cofactors involved in 1-C transfers in amino acid metabolism in more detail: tetrahydrofolate (FH₄) and SAM.

Tetrahydrofolate (FH₄)

Tetrahydrofolate (FH₄) is a key cofactor in the metabolism of amino acids and the biosynthesis of nucleotides. It is also commonly abbreviated as THF, especially in the names of enzymes that use it. To avoid confusion with chemists' use of THF for tetrahydrofuran, we will refer to it mostly as FH₄; however, we will name the enzymes as derivatives of the abbreviation XHF (X = D for dihydro and T for tetrahydro).

FH₄ carries 1C units in various oxidation states. It receives 1C units and then transfers them to other species. The structure of FH₄ and derivatives carrying 1C units with different oxidation numbers (indicating their oxidation state) are shown in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): 1C derivatives in different oxidation states for tetrahydrofolate

FH₄ is made from the vitamin folate (bacteria, fungi, and most plants can synthesize it), which is first converted to dihydrofolate (FH₂) and then tetrahydrofolate (FH₄) by the enzyme dihydrofolate reductase, using NADPH as a reducing substrate "cofactor." The model below depicts this small enzyme complexed with NADPH (NADP⁺), folate, and DHFR (7dfr). Folate (FOL) is shown in salmon spacefill, while NADPH is shown in cyan.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of dihydrofolate reductase with bound NADPH (NADP⁺) and folate (7dfr).

Figure \(\PageIndex{7}\): Dihydrofolate reductase with bound NADP⁺ and folate (7dfr). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ojdpBZuQH8Lm96

Folate (FOL) and NADP⁺ (NAP) are shown in spacefill and CPK colors. Asp 27 side chain is shown in sticks and CPK colors. The oxygen atoms of two water molecules interacting with folate are shown as red spheres. A disordered and, hence, mobile loop (residues 16-20) that binds the nicotinamide is shown in cyan.​

Because FH₄ is a key substrate in many reactions, including nucleotide synthesis (as we shall see later), this enzyme is a key chemotherapy target to kill cancer cells, which require robust nucleotide synthesis for rapid proliferation. One such drug, which inhibits the enzyme, methotrexate, is shown in Figure \(\PageIndex{8}\).

Figure \(\PageIndex{8}\): A structural comparison of the chemotherapeutic drug methotrexate and folate

Before getting into the nitty-gritty of amino acid degradation and tetrahydrofolate chemistry, let's take a look at the mechanism by which dihydrofolate reductase, DHFR, catalyzes the sequential reduction by 2 NADPH molecules of folate to tetrahydrofolate. This is illustrated in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\): Conversion of folate to tetrahydrofolate by dihydrofolate reductase

One Carbon Chemistry: The Interconversion of FH₄ 1C derivatives

Tetrahydrofolate carries 1C units in metabolism. As such, it is intimately involved in many anabolic and catabolic reactions. These include thymidine and purine biosynthesis and amino acid metabolism through reactions involving serine, glycine (both with 1C in their side chains), and methionine (with 1C after the sulfur in the side chain). Reactions occur in both the cytoplasm and mitochondria. FH₄ is directly or indirectly involved in epigenetic control of gene expression, as methylation of both DNA and histones is critical for gene expression. The myriad 1C derivatives of FH₄ make the biochemistry complex.  Because 1C transfer is a critical step in the breakdown of amino acid carbon skeletons and biosynthesis, we need to explore it. The complexity is simplified by noting that the 1C derivatives have only three different oxidation states (+2, 0, and -2), as noted in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Interconversion of 1C derivatives of tetrahydrofolate. The oxidation number of the 1C addition is shown in red. (after http://education.med.nyu.edu/mbm/aminoAcids/folate.shtml)

1C units usually enter FH₄ as the N⁵,N¹⁰ methylene unit. This occurs in the cytoplasm and mitochondria because 1C-derivatized FH₄ does not appear to cross mitochondrial membranes. The methylene derivative (with a 1C oxidation # = 0) can be reduced to form methyl (oxidation # -2) or oxidized to methenyl or formyl groups (oxidation # +2).

We will revisit some of these reactions in chapters on amino acid and nucleotide biosynthesis. That's not a bad thing—learning occurs best when material is repeated in different contexts.

Methylcobalamin and ⁵N-methyl FH₄

Methyl groups can be stored and transferred from ⁵N-methyl FH₄ to another cofactor, cobalamin, to form methylcobalamin (Vitamin B12).  The -CH₃ can then be transferred to homocysteine  (the side chain is the same as for Cys but with one extra -CH₂ methylene group) to form methionine.  This adds to the complexity of 1C transfer, but the iCn3D models below should help you to differentiate the chemical structures of each.

Here is a ball-and-stick model of methylcobalamin (vitamin B12).

Note that the -CH₃ is connected to the central cobalt in the macrocyclic ring.

In contrast, here is the structure of N⁵-methyl FH₄.

The -CH₃ here is attached to the N5 ring position of FH₄.

Serine Hydroxymethyltransferase (SHMT): A complex reaction needs two cofactors - PLP and FH₄

Let's examine one mechanism that illustrates how a 1C methylene is added to tetrahydrofolate (FH₄). The mechanism shown is for serine dehydratase, also known as serine hydroxymethyltransferase. The enzyme not only uses tetrahydrofolate as a substrate but also PLP, which, as we have seen previously, forms bonds to the alpha-carbon of amino acids that are labile to cleavage. In this case, serine dehydrates through an alpha-elimination reaction. Here is the overall reaction.

Serine + FH₄ ↔ Glycine + N⁵,N¹⁰-CH₂-FH₄ + H₂O

Figure \(\PageIndex{11}\) shows the dehydration reaction and formation of glycine, using PLP as a cofactor. Figure B shows how the released formaldehyde reacts with FH₄ to form N⁵,N¹⁰-methylene FH4, using FH₄ as a cofactor. Because this reaction is needed for purine and thymidylate synthesis, SHMT is also a target for malaria treatment.

Figure \(\PageIndex{11}\): Serine hydroxymethyltransferase reaction mechanism

Glu 57 plays a key role as a general acid/base throughout the enzyme's catalytic cycle.

Pathway diagrams showing a myriad of reactants, products, and enzymes can be very confusing. It's helpful to think about them and see them in different ways. Here is another way to present the conversion of FH₄ and its various 1C intermediates. Figure \(\PageIndex{12}\) concentrates on the outputs of 1C FH₄ derivatives (shown in blue ellipses).

Figure \(\PageIndex{12}\): Folate 1C Metabolism - Cyclic Presentation after Ducker and Rabinowitz, Cell Metab. 2017 January 10; 25(1): 27–42. doi:10.1016/j.cmet.2016.08.009

The table below shows the enzymes that catalyze these reactions. This figure applies to both catabolic and anabolic reactions using FH₄ derivatives. Oxidation numbers for the 1C adducts are shown. Any reaction that changes the redox state must use NAD(P)⁺/NAD(P)H as a redox reagent.

• N⁵,N¹⁰-methylene FH₄ gives thymidine and serine
• N⁵-methyl FH₄ gives methionine
• N¹⁰-formyl FH₄ gives formate, purines, and CO₂
• N⁵-formyl serves more as a passive reservoir of 1C units.

Table \(\PageIndex{1}\): Enzymes catalyzing interconversions of THF derivatives

Abbreviation	Enzyme Name
ALDH	10-formyltetrahydrofolate((aldehyde) dehydrogenase.
DHFR	Dihydrofolate reductase
MTHFD	methylenetetrahydrofolate dehydrogenase
MTHFD1	C-1-tetrahydrofolate synthase, cytoplasmic
MTHFD1L	monofunctional tetrahydrofolate synthase, mitochondrial
MTHFD2/L	methylenetetrahydrofolate dehydrogenase 2/2-like
MTFMT	mitochondrial methionyl-tRNA formyltransferase
MTHFR	methylenetetrahydrofolate reductase
MTR	methionine synthase
TYMS	thymidylate synthetase

A key enzyme in these reactions, methylene-THF reductase (MTHFR), irreversibly removes 1C from the cycle depicted above, forming N⁵-methyl FH₄. As this reaction is a reduction, it requires a reducing agent (NADPH in yeast and animals and NADH in plants). As we will see next, this irreversible removal would deplete the cycle shown; therefore, another methylating agent, S-adenosylmethionine (SAM, also known as adoMet), allosterically regulates (inhibits) it. Plant versions of the enzyme are reversible, so SAM does not need to regulate them in this feedback loop process.

S-adenosylmethionine (SAM), also known as AdoMet

N⁵-methyl FH₄ appears to have one function: to methylate a molecule called homocysteine (same as Cys but with an extra -CH₂ in the side chain) to methionine. This is adenosylated (not phosphorylated!) with ATP to produce S-adenosylmethionine (SAM), a more potent methylating agent than N⁵-methyl FH₄. SAM is, hence, part of a cycle involving N5-methyl-FH4. The methyl group of N⁵-methyl FH₄ reacts with homocysteine to produce methionine, catalyzed by the enzyme methionine synthase, which requires cobalamin (vitamin B12) as a cofactor. The combined Figure \(\PageIndex{13}\) adds the Met and Folate cycles (showing the outputs of the 1C Folate metabolism cycle.

Figure \(\PageIndex{13}\): Folate and Methionine Cycle (after Ducker and Rabinowitz, Cell Metab. 2017 Jan 10; 25(1): 27–42.)

Figure \(\PageIndex{14}\) shows the structures of molecules in the Met Cycle.

Figure \(\PageIndex{14}\): The Met Cycle and Methylations by SAM

The mechanism of methyl transfer using SAM as the -CH₃ donor involves an SN₂ attack by a nucleophile of the substrate on the CH₃ of SAM, with the electron pair from the C-S bond going to the positively charged sulfonium sulfur, a strong "electron sink". An analogous nucleophilic attack on the terminal CH₃ of plain old methionine would not be readily enabled. Hence, SAM, with its charged S, has a much higher methyl transfer potential than N⁵-CH₃-FH₄.

In the actual reaction catalyzed by methionine synthase (MS) in mammals, the methyl-CH₃ from N⁵-methyl FH₄ is first transferred to cobalamin, a derivative of vitamin B12, to form methylcobalamin, which then transfers it to homocysteine. The structure of the C-terminal half of B₁₂-dependent Methionine Synthase from E. Coli with bound adenosylhomocysteine (3iva) is shown below and in this link:

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the C-terminal half of B₁₂-dependent Methionine Synthase from E. Coli with bound adenosylhomocysteine bound (3iva)

Figure \(\PageIndex{15}\): C-terminal half of B₁₂-dependent Methionine Synthase from E. Coli with bound adenosylhomocysteine (3iva). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...E7jTsdvVvx1M1A

In mammals, vitamin B₁₂ is a key cofactor in only two enzymes.  One is methionine synthase. The other catalyzes the conversion of methylmalonyl-CoA (derived from the metabolism of Ile, Thr, Val, and Met (as we will see in the next chapter section) to succinyl-CoA in the citric acid cycle. If vitamin B₁₂ is lacking, N⁵-methyl FH₄ builds up when it can't be transferred to cobalamin to form methylcobalamin, the direct methylating agent in methionine synthesis catalyzed by methionine synthase.  As a result, homocysteine builds up because it can't be converted to methionine. Since the enzyme that converts N⁵,N¹⁰-methylene FH₄ to N⁵-methyl FH₄, methylenetetrahydrofolate reductase (MTHFR), is irreversible, levels of N⁵-methyl FH₄ are high. You can view N⁵-methyl FH₄ as a storage form of folate, so if there is a lack of B12, the use of this storage form of folate becomes impaired.  B12 deficiency can cause megaloblastic anemia.

The Serine Glycine One Carbon (SGOC) Metabolic Cycle

Figure 12 illustrates the coupled Folate and Methionine cycles, highlighting the intermediates involved in the 1C-CH₃ transfer reaction, a crucial aspect of amino acid metabolism and other anabolic and catabolic processes. Interpreting metabolic figures is complicated. Each is designed to emphasize certain selected features. Another way to present Figure 12 is to emphasize the metabolites involved in 1C chemistry. Figure \(\PageIndex{145}\) shows what has been called the Serine Glycine One Carbon (SGOC) metabolic cycle. It is simply a redrawn version of Figure 12, with attention drawn to non-FH4 molecules involved in 1C transfers, specifically serine, glycine, and formate.

Figure \(\PageIndex{15}\): The Ser-Gly One Carbon (SGOC) Cycle

This cycle, along with its key substrates serine and glycine, integrates numerous metabolic pathways and regulates the conversion of serine and glycine into essential outputs for other pathways. We will see this cycle again in the chapter on amino acid biosynthesis. The pathway is especially important in tumor cells, which need precursors for nucleic acid, protein, and lipid synthesis.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter describes the metabolic fates of amino acid carbon skeletons after deamination and introduces the biochemistry of one-carbon (1C) transfer reactions mediated by tetrahydrofolate (FH4) and S-adenosylmethionine (SAM), two cofactors central to amino acid catabolism, nucleotide biosynthesis, and epigenetic regulation.

After transamination and oxidative deamination remove the amino group, the remaining carbon skeletons are converted to metabolic intermediates that enter either glycolysis or the TCA cycle. Amino acids are classified by the entry points their skeletons occupy: purely ketogenic amino acids (only leucine and lysine) yield acetyl-CoA or acetoacetyl-CoA, which can enter the TCA cycle but cannot produce net glucose because the pyruvate dehydrogenase reaction is irreversible and two carbons are lost as CO₂ per turn of the cycle. Purely glucogenic amino acids yield pyruvate or direct TCA cycle intermediates (α-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate) that can support net glucose synthesis through gluconeogenesis; their TCA cycle entry reactions are anaplerotic, replenishing the pool of cycle intermediates. A structural heuristic helps predict classification: the number of carbons in the longest continuous chain of the amino acid generally matches the number of carbons in the TCA intermediate it produces (e.g., five-carbon glutamate → five-carbon α-ketoglutarate; four-carbon aspartate → four-carbon oxaloacetate; three-carbon alanine → three-carbon pyruvate). Amino acids with long or fat-like side chains (Leu, Ile, Lys, Phe, Tyr, Trp) tend to be fully or partly ketogenic. Five amino acids — Trp, Tyr, Phe, Ile, and Thr — are both glucogenic and ketogenic, yielding products at multiple oxidation states.

A wide variety of enzyme cofactors facilitate the breaking, formation, and rearrangement of bonds in amino acid carbon skeletons. These include all previously encountered cofactors (PLP, TPP, biotin, NAD⁺/NADP⁺, FAD, coenzyme A) as well as two particularly important 1C carriers: tetrahydrofolate (FH₄) and S-adenosylmethionine (SAM). FH₄ is derived from the vitamin folate through two sequential NADPH-dependent reductions catalyzed by dihydrofolate reductase (DHFR). Because FH₄ is essential for thymidylate and purine synthesis — and hence for DNA replication in rapidly dividing cells — DHFR is a premier target for anticancer drugs. Methotrexate, a structural analog of folate, competitively inhibits DHFR and depletes the cellular pool of FH₄, blocking nucleotide biosynthesis and killing proliferating cells.

FH₄ functions as a carrier of one-carbon units in three oxidation states. The most oxidized form (oxidation state +2) is carried as N¹⁰-formyl-FH₄ or N⁵,N¹⁰-methenyl-FH₄ and contributes carbons to purine ring synthesis and formate production. The intermediate oxidation state (0) is the N⁵,N¹⁰-methylene-FH₄, which is used in thymidylate synthesis (providing the methyl group to dUMP to form dTMP, catalyzed by thymidylate synthase) and in serine biosynthesis. The most reduced form (−2) is N⁵-methyl-FH₄, whose sole known function is to donate its methyl group to homocysteine to form methionine. The interconversion of these derivatives requires NAD(P)H for reductive steps and releases NAD(P)H in oxidative steps, integrating 1C metabolism with cellular redox state.

The entry of 1C units into the folate pool is exemplified by serine hydroxymethyltransferase (SHMT), which catalyzes the reversible conversion of serine and FH₄ to glycine and N⁵,N¹⁰-methylene-FH₄. The reaction uses two cofactors simultaneously: PLP facilitates α-elimination of the serine side chain via its standard carbanion-stabilizing mechanism, and FH4 captures the released formaldehyde by nucleophilic attack, forming the methylene bridge. Because SHMT is indispensable for nucleotide synthesis in rapidly proliferating parasites and cancer cells, it is a target for antimalarial therapy.

The methionine cycle is closely coupled to folate 1C metabolism. N⁵-methyl-FH₄transfers its methyl group to cobalamin (vitamin B12) to form methylcobalamin, which then methylates homocysteine in a reaction catalyzed by methionine synthase, producing methionine and regenerating FH₄. Methionine is then adenosylated by ATP — uniquely, via attack on the adenosyl group rather than phosphoryl transfer — to form S-adenosylmethionine (SAM). SAM is the principal biological methyl donor: its positively charged sulfonium group serves as an excellent electron sink, making it far more reactive toward nucleophilic S_N2 attack than N⁵-methyl-FH₄whose methyl group is on a neutral nitrogen. SAM methylates DNA, histones, phospholipids, neurotransmitters, and many other substrates. After methyl transfer, SAM is converted to S-adenosylhomocysteine, which is hydrolyzed to homocysteine, completing the cycle. The enzyme methylenetetrahydrofolate reductase (MTHFR), which irreversibly converts N⁵,N¹⁰-methylene-FH₄ to N⁵-methyl-FH₄, is allosterically inhibited by SAM — a feedback mechanism that prevents the 1C pool from being irreversibly drained into the methyl form when methylating capacity is already sufficient. Vitamin B12 deficiency traps folate as N⁵-methyl-FH4 (the "methyl trap"), depleting other FH₄ derivatives and impairing thymidylate and purine synthesis, resulting in megaloblastic anemia due to impaired erythrocyte DNA replication. The Serine-Glycine One Carbon (SGOC) cycle integrates all of these reactions into a unified framework, showing how serine, glycine, and formate serve as the primary 1C unit donors that fuel nucleotide synthesis, methylation reactions, and redox balance — particularly in tumor cells, which upregulate SGOC flux to meet their elevated demands for nucleic acid and lipid biosynthesis.