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18.4: An overview of amino acid metabolism and the role of Cofactors

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    15034
  • Amino acid degradation

    We saw how nitrogen is removed from amino acids to produce urea or NH4+ in the previous chapter section. What are the fates of the carbon skeletons that remain? This section is where student might get overwhelmed by the diversity of amino acid degradation pathways, so it helps to realize that 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 daunting but also fascinating, as organisms try to extract all the energy and molecule-building atoms from a metabolite and minimize waste.

    Here are some key features of amino acid catabolism that chapter section will present.

    • some are converted to pyruvate, the end product of glycolysis and the start reactant of gluconeogenesis. Hence, these amino acids are glucogenic;
    • some are converted to acetoacetate-CoA and or acetyl-CoA. Both of these can be converted to ketone bodies (acetoacetate/β-hydroxybutyrate) so these are considered ketogenic. Since the two carbons of the acetyl group of acetyl-CoA are lost as CO2 in the TCA cycle, and their 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 net fashion;
    • some are metabolized to form TCA intermediates. Since they are added in net fashion to the TCA cycle and don't remove the existing pool of TCA intermediates, they can produce in net fashion direct or indirectly molecules that can be use to produce glucose. These entry reactions to the TCA which replenish or add to TCA intermediates are called anerplerotic (replenishing) reactions. Hence these amino acid are also glucogenic.
    • some have multiple ways to be degraded and can produce both acetyl-CoA and pyruvate, so they are both glucogneic and ketogenic.

    Let's get more explicit:

    • purely ketogenic: only Leu and Lys (the only amino acids whose name starts with L and you have to Love them since there are only 2 amino acids in this category)
    • both: 5 are including the aromatics - Trp, Tyr, Phe - and Ile/Thr
    • purely glucogenic: the rest

    General hints as to if an amino acid is glucogenic and/or ketogenic can be derived from their structures.

    Fully or Partly Ketogenic Amino Acids: Amino acids with the exception of 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/glucogeneic. These include Lys, Leu, Ile, and the aromatic amino acids Phe, Trp and Tyr and exclude Val. (note: Thr, which is both ketogenic and glucogenic, doesn't fit this rule).

    KetogenicFatLike.png

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

    Purely Glucogenic Amino Acids (for Pyr and/or TCA intermediates: Which amino acids produces which intermediate. With the exception of 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)

    GlucogeneicAAto5C_aKG.png

    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 iin the continuous chain produced oxalacetate (which is more oxidized than succinate), while the least oxidized ones produce succinate acid (which is more reduced than oxaloacetate)

    GlucogeneicAAto4C_succOROAA.png

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

    These form for the 3C amino acids, Ser, Ala and Cys, along with the exception Glycine

    GlucogeneicAAto3C_Pyrvate.png

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

    Here is a full figure summarizing everything above!

    summaryAACsTCACyleKetoGluogeneic.png

    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 of carbon atoms in amino acid catabolism is daunting. As with all reactions, a pathway for a flow of electrons and stabilization of transition states and intermediates must be in place for the reactions to occur. All methods of catalysis (general acid/base, covalent/nucleophilic catalysis, electrostatic/metal ion catalysis, preferential stabilization of the transition state occur. Yet some reaction need addition "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. We've see the important role of pyridoxal phosphate in transaminations/amino transferases in the previous section. 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 once bound usually stay put. That is, they don't dissociate during the catalytic cycle. For instance, PLP must initial bind to an enyzme, but then becomes covalently attached through a Schiff base through the epsilon amino group of a Lys in the catalytic site. That linkage might swap with an incoming amino acid substrate, for examples, but reforms in the catalytic cycle so the enzyme remains functional.
    • coenzyme - a vitamin derivative organic cofactor, as compared to an inorganic ions, for example. Coenzyme is a poor historical name that still persists.
    • prosthetic group - this is usually a coenzyme that is either covalently bound (like PLP) or noncovalently bound with a such a low KD (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 nonmetalic 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

    Examples of other nonvitamin cofactors include S-adenosylmethione (SAM or adoMet), a carrier of methyl groups, coenzyme Q, a carrier of electrons, tetrahydrobiopterin, a carrier of oxygen and electrons, and of course heme, a carrier of electrons.

    We've already described amino acid deaminations 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 metabolism of amino acids but it is also critical in the biosynthesis of nucleotides. It is also commonly abbreviated as THF, especially in the names of enzymes that use it. To avoid confusion with chemist's use of THF for tetrahydrofuran, it will be referred to mostly here as FH4 but the enzymes will be named as derivatives of abbreviation XHF (X = D for dihydro and T for tetrahydro.

    FH4 is a carrier of 1 C units in various oxidation states. It receives 1C units and then transfers them to other species. The structure of FH4 and derivatives carrying 1 C units with different oxidation numbers (indicating their oxidation state) are shown below.

    tetrahydrofolate_derivatives.png

    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 gets converted first to dihydrofolate (FH2) then tetrahydrofolate (FH4), using NADPH as a reducing substrate "cofactor", by the enzyme dihydrofolate reductase. The model below shows this small enzyme with NADPH (NADP+) and folate bound to DHFR (7dfr). Folate (FOL) is shown in salmon space fill while NADPH is shown in cyan.

    ​(external link to structure) https://structure.ncbi.nlm.nih.gov/i...2oWvZfP4awoEu7

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

    folate_methotrexate.png

    Figure \(\PageIndex{7}\): A structural comparison of the chemotherapeutic drug methotrxate 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.

    folate_tetrahydro_DHFReductase.png

    Figure \(\PageIndex{8}\): Conversion of folate to tetrahydrofolate by dihydrofolate reductase

    Qun Wan, Brad C. Bennett, Mark A. Wilson, Andrey Kovalevsky, Paul Langan, Elizabeth E. Howell, Chris Dealwis

    Proceedings of the National Academy of Sciences Dec 2014, 111 (51) 18225-18230; DOI: 10.1073/pnas.1415856111

    One Carbon Chemistry: The Interconversion of FH4 1C derivatives

    Tetrahydrofolate is a carrier for 1C units in metabolism. As such is it intimately involved in many anabolic and catabolic reactions. These include thymidine and purine biosynthesis and amino acids metabolism through reactions involving serine, glycine (both with 1 C in their side chains) and methionine (with 1C after the sulfur in the side chain). The reactions takes place in both the cytoplasm and mitochondria. FH4 is involved directly or indirectly in epigenetic control of DNA expression as well, as methylation of both DNA and histones is critical to gene expression. The myriad of 1C derivatives of FH4 make the biochemistry complex, but as the transfer of 1C is a critical job in both the breakdown of the carbon skeleton of amino acids and in 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). We will see some of these reaction again in chapters dealing with amino acid and nucleotide biosynthesis. That's not a bad thing - learning occurs best on repetition of material in different contexts.

    tetrahydrofolate_derivatives_interconversions.png

    Figure \(\PageIndex{9}\): Interconversion of 1C derivatives of tetrahydrofolate. Oxidation number of the 1C addition are shown in red.

    (after http://education.med.nyu.edu/mbm/aminoAcids/folate.shtml0

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

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

    Let's look in detail at one mechanism that shows how a 1C methylene is added to tetrahydrofolate (FH4). The mechanism shown is for serine dehydratase, aka serine hydroxymethyltransferase. The enzyme not only uses tetrahydrofolate as a substrate but also PLP, which, as we have seen previously, makes bonds to the alpha-carbon of amino acids labile to cleavage. In this case, the amino acid serine becomes dehydated through an alpha elimination reaction. Here is the overall reaction.

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

    Figure A below 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. As this is needed in purine and thymidylate synthesis, SHMT is a target for malaria treatment as well.

    SerineDehydratasePLPTHFRx.png
    Figure \(\PageIndex{10}\): Serine hydroxymethylltransferase reaction mechanism

    Glu 57 plays a key role as a general acid/based throughout the catalytic cycle of the enzyme.

    Pathway diagrams showing a myriad of reactants, products and enzymes can be really confusing to students (and to authors as well). It's useful 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. This figure below concentrates on the outputs of 1C FH4 derivatives (shown in blue eclipses). The enzymes catalyzing this reactions are shown in the table below. This figure applies to both catabolic and anabolic reactions using FH4 derivatives. Oxidation numbers for the 1C adducts are shown. Any reaction that involves a change in redox state must use NAD(P)+/NAD(P)H as 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.

    CYCLE_tetrahydrofolate_interconversions.png

    Figure \(\PageIndex{11}\): 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

    Table:
    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 as it forms N5-methyl FH4. As this reaction is a reduction, it requires a reducing agent (NADPH in yeast and animals and NADH in plants). This irreverisble removal would deplete the cycle shown so it is allosterically regulated (inhibited) by another methylating agent, S-adenosylmethionine (SAM aka adoMet), as we will see next. Plant versions of the enzyme ae reversible so there is no need for regulation by SAM in this feedback loop process.

    S-adenosylmethionine (SAM aka 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 phosphoyrlated!) 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 figure below adds Figure 11 (showing the outputs of the 1C Folate metabolism cycle

    SAM_MethylTHF.png

    Figure \(\PageIndex{12}\): Folate and Methionine Cycle

    after Ducker and Rabinowitz, Cell Metab. 2017 Jan 10; 25(1): 27–42.

    The figure below shows the structures of molecules in the Met Cycle.

    Strucutres_SAM_MethylTHF.png

    Figure \(\PageIndex{13}\): The Met Cycle and Methylations by SAM

    The mechanism of methyl transfers using SAM as the -CH3 donor involves a 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 sulfure, a great "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 high 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 B6, 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 adenoslylhomocysteine bound (3iva) is shown below and in this link:

    https://structure.ncbi.nlm.nih.gov/i...2sNHQNCbtGy9F7

    In mammals, vitamin B12 is a key cofactor in only two enzymes, on being methionine synthase. If vitamin B12 is lacking, N5-methyl FH4 builds up and since the enzyme that converts N5,N10-methylene FH4 to N5-methyl FH4, methylenetetrahydrofolate reductase (MTHFR), is irreversible. This leads to megaloblastic anemia as precursors to red blood cells can't mature. Folate deficiencies also lead to anemia.

    The Serine Glycine One Carbon (SGOC) Metabolic Cycle

    Figure 12 shows the coupled Folate and Methionine cycles that emphasize the intermediates involved in the 1C -CH3 transfer reaction, an important part of amino acid metabolism but other anabolic and catabolic reactions as well. Interpreting metabolic figures is complicated. Each is designed to emphasis certain selected features. Another way to present Figure 12 is to emphasize metabolites involved in 1C chemistry in general. The figure below show what has been called the Serine Glycine One Carbon (SGOC) metabolic cycle. It is just a redrawn version of Figure 12 with attention drawn to non FH4 molecules involved in 1C transfers, namely serine, glycine and also formate.

    SerGly1CCycle.png

    Figure \(\PageIndex{14}\): The Ser-Gly One Carbon (SGOC) Cycle

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

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