Acetoacetate decarboxylase

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Acetoacetate decarboxylase
Acetoacetate decarboxylase biounit 3BH3 with inset.png
Acetoacetate decarboxylase dodecamer structure with bound 2-Pentanone bound in its active sites.
Identifiers
EC no. 4.1.1.4
CAS no. 9025-03-0
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Acetoacetate decarboxylase (AAD or ADC) is an enzyme (EC 4.1.1.4) involved in both the ketone body production pathway in humans and other mammals, and solventogenesis in bacteria. Acetoacetate decarboxylase plays a key role in solvent production by catalyzing the decarboxylation of acetoacetate, yielding acetone and carbon dioxide. [1]

Contents

This enzyme has been of particular interest because it is a classic example of how pKa values of ionizable groups in the enzyme active site can be significantly perturbed. Specifically, the pKa value of lysine 115 in the active site is unusually low, allowing for the formation of a Schiff base intermediate and catalysis. [2]

acetoacetic acid Acetoacetate decarboxylase acetone
Acetoacetic acid.png   Acetone-2D-skeletal.svg
CO2
Biochem reaction arrow forward NYNN horiz med.svg
 
 

History

Acetoacetate decarboxylase is an enzyme with major historical implications, specifically in World War I and in establishing the state of Israel. [3] During the war the Allies needed pure acetone as a solvent for nitrocellulose, a highly flammable compound that is the main component in gunpowder. [4] In 1916, biochemist and future first president of Israel Chaim Weizmann was the first to isolate Clostridium acetobutylicum, a Gram-positive, anaerobic bacteria in which acetoacetate decarboxylase is found. Weizmann was able to harness the organism's ability to yield acetone from starch in order to mass-produce explosives during the war. [3] This led the American and British governments to install the process devised by Chaim Weizmann in several large plants in England, France, Canada, and the United States. Through Weizmann's scientific contributions in World War I, he became close with influential British leaders educating them of his Zionist beliefs. [5] One of them was Arthur Balfour, the man after whom the Balfour Declaration—the first document pronouncing British support in the establishment of a Jewish homeland—was named.

The production of acetone by acetoacetate decarboxylase-containing or clostridial bacteria was utilized in large-scale industrial syntheses in the first half of the twentieth century. In the 1960s, the industry replaced this process with less expensive, more efficient chemical syntheses of acetone from petroleum and petroleum derivatives. [6] However, there has been a growing interest in acetone production that is more environmentally friendly, causing a resurgence in utilizing acetoacetate decarboxylase-containing bacteria. [7] Similarly, isopropanol and butanol fermentation using clostridial species is also becoming popular.

Structure

Crystal structure of acetoacetate decarboxylase in complex with an inhibitor, pentan-2-one. Using the residue Lys115, this inhibitor forms an acetyl acetone Schiff base intermediate. Mostly hydrophobic residues, such as Met 97 and Phe27, are found in the active site. This image was generated using PyMOL, from PDB ID 3BH3. AAD active site.png
Crystal structure of acetoacetate decarboxylase in complex with an inhibitor, pentan-2-one. Using the residue Lys115, this inhibitor forms an acetyl acetone Schiff base intermediate. Mostly hydrophobic residues, such as Met 97 and Phe27, are found in the active site. This image was generated using PyMOL, from PDB ID 3BH3.

Acetoacetate decarboxylase is a 365 kDa complex with a homododecameric structure. [8] The overall structure consists of antiparallel β-sheets and a central seven-stranded cone-shaped β-barrel. The core of this β-barrel surrounds the active site in each protomer of the enzyme. The active site, consisting of residues such as Phe27, Met97, and Tyr113, is mostly hydrophobic. However, the active site does contain two charged residues: Arg29 and Glu76.

Arg29 is thought to play a role in substrate binding, while Glu76 is thought to play a role in the orienting the active site for catalysis. The overall hydrophobic environment of the active site plays a critical role in favoring the neutral amine form of Lys115, a key residue involved in the formation of a Schiff base intermediate. Another important lysine residue, Lys116, is thought to play an important role in the positioning of Lys115 in the active site. Through hydrogen bonds with Ser16 and Met210, Lys116 positions Lys115 in the hydrophobic pocket of the active site to favor the neutral amine form.

Reaction mechanism

Figure 1: General mechanism of acetoacetate decarboxylase, proceeding through a Schiff base intermediate and producing acetone and carbon dioxide AAD final mech.jpg
Figure 1: General mechanism of acetoacetate decarboxylase, proceeding through a Schiff base intermediate and producing acetone and carbon dioxide

Acetoacetate decarboxylase from Clostridium acetobutylicum catalyzes the decarboxylation of acetoacetate to yield acetone and carbon dioxide (Figure 1). The reaction mechanism proceeds via the formation of a Schiff base intermediate, which is covalently attached to lysine 115 in the active site. The first line of support for this mechanism came from a radiolabeling experiment in which researchers labeled the carbonyl group of acetoacetate with 18O and observed that oxygen exchange to water, used as the solvent, is a necessary part of decarboxylation step. [9] These results provided support that the mechanism proceeds through a Schiff base intermediate between the ketoacid and an amino acid residue on the enzyme.

Further research led to the isolation of an active site peptide sequence and identification of the active site lysine, Lys115, that is involved in the formation of the Schiff base intermediate. [2] [10] Additionally, later experiments led to the finding that maximum activity of the enzyme occurs at pH 5.95, suggesting that the pKa of the ε-ammonium group of Lys115 is significantly perturbed in the active site. [2] If the pKa were not perturbed downward, the lysine residue would remain protonated as an ammonium cation, making it unreactive for the nucleophilic addition necessary to form the Schiff base.

Figure 2: Scheme of the reaction, with reporter molecule 5-NSA, used by Westheimer et al. to measure the pKa of Lys115 in the active site. Reporter molecule scheme Westheimer.jpg
Figure 2: Scheme of the reaction, with reporter molecule 5-NSA, used by Westheimer et al. to measure the pKa of Lys115 in the active site.

Building upon this finding, Westheimer et al. directly measured the pKa of Lys115 in the active site using 5-nitrosalicylaldehyde (5-NSA) . Reaction of 5-NSA with acetoacetate decarboxylase and subsequent reduction of the resulting Schiff base with sodium borohydride led to the incorporation of a 2-hydroxy-5-nitrobenzylamino reporter molecule in the active site (Figure 2). Titration of the enzyme with this attached reporter group revealed that the pKa of Lys115 is decreased to 5.9 in the active site. [12] These results were the basis for the proposal that the perturbation in the pKa of Lys115 was due to its proximity to the positively charged ε-ammonium group of Lys116 in the active site. [2] A nearby positive charge could cause unfavorable electrostatic repulsions that weaken the N-H bond of Lys115. Westheimer et al.'s proposal was further supported by site-directed mutagenesis studies. When Lys116 was mutated to cysteine or asparagine, the pKa of Lys115 was found to be significantly elevated to over 9.2, indicating that positively charged Lys116 plays a critical role in determining the pKa of Lys115. [2] Although a crystal structure was not yet solved to provide structural evidence, this proposal was widely accepted and cited as a textbook example of how the active site can be precisely organized to perturb a pKa and affect reactivity. [8]

In 2009, a crystal structure of acetoacetate decarboxylase from Clostridium acetobutylicum was solved, allowing Westheimer et al.'s proposal to be evaluated from a new perspective . From the crystal structure, researchers found that Lys 115 and Lys 116 are oriented in opposite directions and separated by 14.8 Å (Figure 3). [8] This distance makes it unlikely that the positive charge of Lys116 is able to affect the pKa of Lys115. Instead through hydrogen bonds with Ser16 and Met210, Lys116 likely holds Lys115 into position in a hydrophobic pocket of the active site. This positioning disrupts the stability of the protonated ammonium cation of Lys115, suggesting that the perturbation of Lys115's pKa occurs through a 'desolvation effect'.

Figure 3: Orientation of Lysine 115 and 116. In the active site, lysine 115 and 116 point away from each other and are separated by 14.8 A. This image was generated using PyMOL, from PDB ID 3BH3. Lysines in active site.png
Figure 3: Orientation of Lysine 115 and 116. In the active site, lysine 115 and 116 point away from each other and are separated by 14.8 Å. This image was generated using PyMOL, from PDB ID 3BH3.

Inactivation and inhibition

Acetoacetate decarboxylase is inhibited by a number of compounds. Acetic anhydride performs an electrophilic attack on the critical catalytic residue, Lys115, of acetoacetate decarboxylase to inactivate the enzyme. [13] The rate of inactivation was assessed through the hydrolysis of the synthetic substrate 2,4-dinitrophenyl propionate to dinitrophenol by acetoacetate decarboxylase. In the presence of acetic anhydride, the enzyme is inactivated, unable to catalyze the hydrolysis reaction 2,4-dinitrophenyl propionate to dinitrophenol. [14]

Acetonylsulfonate acts as a competitive inhibitor (KI=8.0 mM ) as it mimics the characteristics of the natural substrate, acetoacetate (KM=8.0 mM). [15] The monoanion version of acetonylphosphonate is also a good inhibitor (KI=0.8mM), more efficient than the acetonylphosphonate monoester or dianion. [16] These findings indicate that active site is very discriminatory and sterically restricted.

Hydrogen cyanide seems to be an uncompetitive inhibitor, combining with Schiff's base compounds formed at the active site. [15] Addition of carbonyl compounds to the enzyme, in the presence of hydrogen cyanide, increases hydrogen cyanide's ability to inhibit acetoacetate decarboxylase, suggesting that carbonyl compounds readily form Schiff's bases at the active site. Hydrogen cyanide is most potent as an inhibitor at pH 6, the optimum pH for the enzyme, suggesting that the rate-limiting step of catalysis is the formation of the Schiff base intermediate.

Beta-diketones appear to inhibit acetoacetate decarboxylase well but slowly. Acetoacetate decarboxylase has a KM for acetoacetate of 7×10−3 M whereas the enzyme has a KI for benzoylacetone of 1.9×10−6 M. [15] An enamine is most likely formed upon interaction of beta-diketones with free enzyme.

The reaction of acetoacetate decarboxylase with p-chloromercuriphenylsulfonate (CMS) results in decreased catalytic activity upon two equivalents of CMS per enzyme subunit. [15] CMS interacts with two sulfhydryl groups located on each enzyme subunit. Further inactivation occurs upon addition of a third equivalent of CMS per subunit. Addition of free cysteine to the inhibited enzyme is able to reverse CMS inhibition of acetoacetate decarboxylase.

Activity in bacteria

Acetoacetate decarboxylase has been found and studied in the following bacteria in addition to Clostridium acetobutylicum :

Activity in humans and mammals

While this enzyme has not been purified from human tissue, the activity was shown to be present in human blood serum. [17] [18] The acetoacetate decarboxylase activity is mainly attributed to human serum albumin, however, the activity is about times that of real acetoacetate decarboxylase (C. acetobutylicum). [19]

In humans and other mammals, the conversion of acetoacetate into acetone and carbon dioxide by acetoacetate decarboxylase is a final irreversible step in the ketone-body pathway that supplies the body with a secondary source of energy. [20] In the liver, acetyl co-A formed from fats and lipids are transformed into three ketone bodies: acetoacetate, D-β-hydroxybutyrate and acetone. Acetoacetate and D-β-hydroxybutyrate are exported to non-hepatic tissues, where they are converted back into acetyl-coA and used for fuel. Acetone and carbon dioxide on the other hand are exhaled, and not allowed to accumulate under normal conditions.

Acetoacetate and D-β-hydroxybutyrate freely interconvert through the action of D-β-hydroxybutyrate dehydrogenase. [20] Subsequently, one function of acetoacetate decarboxylase may be to regulate the concentrations of the other, two 4-carbon ketone bodies.

Clinical significance

Ketone body production increases significantly when the rate of glucose metabolism is insufficient in meeting the body's energy needs. Such conditions include high-fat ketogenic diets, diabetic ketoacidosis, or severe starvation. [21]

Under elevated levels of acetoacetate and D-β-hydroxybutyrate, acetoacetate decarboxylase produces significantly more acetone. Acetone is toxic, and can accumulate in the body under these conditions. Elevated levels of acetone in the human breath can be used to diagnose diabetes. [21]

Related Research Articles

<span class="mw-page-title-main">Ketone bodies</span> Chemicals produced during fat metabolism

Ketone bodies are water-soluble molecules or compounds that contain the ketone groups produced from fatty acids by the liver (ketogenesis). Ketone bodies are readily transported into tissues outside the liver, where they are converted into acetyl-CoA —which then enters the citric acid cycle and is oxidized for energy. These liver-derived ketone groups include acetoacetic acid (acetoacetate), beta-hydroxybutyrate, and acetone, a spontaneous breakdown product of acetoacetate.

<span class="mw-page-title-main">Acetoacetic acid</span> Chemical compound

Acetoacetic acid is the organic compound with the formula CH3COCH2COOH. It is the simplest beta-keto acid, and like other members of this class, it is unstable. The methyl and ethyl esters, which are quite stable, are produced on a large scale industrially as precursors to dyes. Acetoacetic acid is a weak acid.

<span class="mw-page-title-main">Acetyl-CoA</span> Chemical compound

Acetyl-CoA is a molecule that participates in many biochemical reactions in protein, carbohydrate and lipid metabolism. Its main function is to deliver the acetyl group to the citric acid cycle to be oxidized for energy production.

<span class="mw-page-title-main">Ketogenesis</span> Chemical synthesis of ketone bodies

Ketogenesis is the biochemical process through which organisms produce ketone bodies by breaking down fatty acids and ketogenic amino acids. The process supplies energy to certain organs, particularly the brain, heart and skeletal muscle, under specific scenarios including fasting, caloric restriction, sleep, or others.

Aromatic <small>L</small>-amino acid decarboxylase Class of enzymes

Aromatic L-amino acid decarboxylase, also known as DOPA decarboxylase (DDC), tryptophan decarboxylase, and 5-hydroxytryptophan decarboxylase, is a lyase enzyme, located in region 7p12.2-p12.1.

<span class="mw-page-title-main">Pyridoxal phosphate</span> Active form of vitamin B6

Pyridoxal phosphate (PLP, pyridoxal 5'-phosphate, P5P), the active form of vitamin B6, is a coenzyme in a variety of enzymatic reactions. The International Union of Biochemistry and Molecular Biology has catalogued more than 140 PLP-dependent activities, corresponding to ~4% of all classified activities. The versatility of PLP arises from its ability to covalently bind the substrate, and then to act as an electrophilic catalyst, thereby stabilizing different types of carbanionic reaction intermediates.

<span class="mw-page-title-main">Isocitrate dehydrogenase</span> Class of enzymes

Isocitrate dehydrogenase (IDH) (EC 1.1.1.42) and (EC 1.1.1.41) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO2. This is a two-step process, which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate. In humans, IDH exists in three isoforms: IDH3 catalyzes the third step of the citric acid cycle while converting NAD+ to NADH in the mitochondria. The isoforms IDH1 and IDH2 catalyze the same reaction outside the context of the citric acid cycle and use NADP+ as a cofactor instead of NAD+. They localize to the cytosol as well as the mitochondrion and peroxisome.

<span class="mw-page-title-main">Pyruvate decarboxylase</span> Class of enzymes

Pyruvate decarboxylase is an enzyme that catalyses the decarboxylation of pyruvic acid to acetaldehyde. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. In anaerobic conditions, this enzyme participates in the fermentation process that occurs in yeast, especially of the genus Saccharomyces, to produce ethanol by fermentation. It is also present in some species of fish where it permits the fish to perform ethanol fermentation when oxygen is scarce. Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide. Pyruvate decarboxylase depends on cofactors thiamine pyrophosphate (TPP) and magnesium. This enzyme should not be mistaken for the unrelated enzyme pyruvate dehydrogenase, an oxidoreductase, that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA.

Acyl-CoA dehydrogenases (ACADs) are a class of enzymes that function to catalyze the initial step in each cycle of fatty acid β-oxidation in the mitochondria of cells. Their action results in the introduction of a trans double-bond between C2 (α) and C3 (β) of the acyl-CoA thioester substrate. Flavin adenine dinucleotide (FAD) is a required co-factor in addition to the presence of an active site glutamate in order for the enzyme to function.

β-Hydroxybutyric acid Chemical compound

β-Hydroxybutyric acid, also known as 3-hydroxybutyric acid or BHB, is an organic compound and a beta hydroxy acid with the chemical formula CH3CH(OH)CH2CO2H; its conjugate base is β-hydroxybutyrate, also known as 3-hydroxybutyrate. β-Hydroxybutyric acid is a chiral compound with two enantiomers: D-β-hydroxybutyric acid and L-β-hydroxybutyric acid. Its oxidized and polymeric derivatives occur widely in nature. In humans, D-β-hydroxybutyric acid is one of two primary endogenous agonists of hydroxycarboxylic acid receptor 2 (HCA2), a Gi/o-coupled G protein-coupled receptor (GPCR).

<span class="mw-page-title-main">Orotidine 5'-phosphate decarboxylase</span>

Orotidine 5′-phosphate decarboxylase or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP). The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate, cytidine triphosphate, and thymidine triphosphate. OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering.

In enzymology, 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is an enzyme that catalyzes the chemical reaction:

<span class="mw-page-title-main">D-lysine 5,6-aminomutase</span>

In enzymology, D-lysine 5,6-aminomutase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Cystathionine beta-lyase</span> Enzyme

Cystathionine beta-lyase, also commonly referred to as CBL or β-cystathionase, is an enzyme that primarily catalyzes the following α,β-elimination reaction

<span class="mw-page-title-main">2-Dehydro-3-deoxy-phosphogluconate aldolase</span> Class of enzymes

The enzyme 2-dehydro-3-deoxy-phosphogluconate aldolase, commonly known as KDPG aldolase, catalyzes the chemical reaction

<span class="mw-page-title-main">Arginine decarboxylase</span>

The enzyme Acid-Induced Arginine Decarboxylase (AdiA), also commonly referred to as arginine decarboxylase, catalyzes the conversion of L-arginine into agmatine and carbon dioxide. The process consumes a proton in the decarboxylation and employs a pyridoxal-5'-phosphate (PLP) cofactor, similar to other enzymes involved in amino acid metabolism, such as ornithine decarboxylase and glutamine decarboxylase. It is found in bacteria and virus, though most research has so far focused on forms of the enzyme in bacteria. During the AdiA catalyzed decarboxylation of arginine, the necessary proton is consumed from the cell cytoplasm which helps to prevent the over-accumulation of protons inside the cell and serves to increase the intracellular pH. Arginine decarboxylase is part of an enzymatic system in Escherichia coli, Salmonella Typhimurium, and methane-producing bacteria Methanococcus jannaschii that makes these organisms acid resistant and allows them to survive under highly acidic medium.

<span class="mw-page-title-main">Diaminopimelate decarboxylase</span> Enzyme decarboxylates diaminopimelate, forming L-lysine

The enzyme diaminopimelate decarboxylase (EC 4.1.1.20) catalyzes the cleavage of carbon-carbon bonds in meso 2,6 diaminoheptanedioate to produce CO2 and L-lysine, the essential amino acid. It employs the cofactor pyridoxal phosphate, also known as PLP, which participates in numerous enzymatic transamination, decarboxylation and deamination reactions.

<span class="mw-page-title-main">Diphosphomevalonate decarboxylase</span> InterPro Family

Diphosphomevalonate decarboxylase (EC 4.1.1.33), most commonly referred to in scientific literature as mevalonate diphosphate decarboxylase, is an enzyme that catalyzes the chemical reaction

Exogenous ketones are a class of ketone bodies that are ingested using nutritional supplements or foods. This class of ketone bodies refers to the three water-soluble ketones. These ketone bodies are produced by interactions between macronutrient availability such as low glucose and high free fatty acids or hormone signaling such as low insulin and high glucagon/cortisol. Under physiological conditions, ketone concentrations can increase due to starvation, ketogenic diets, or prolonged exercise, leading to ketosis. However, with the introduction of exogenous ketone supplements, it is possible to provide a user with an instant supply of ketones even if the body is not within a state of ketosis before ingestion. However, drinking exogenous ketones will not trigger fat burning like a ketogenic diet.

<span class="mw-page-title-main">Coenzyme A transferases</span> Coenzyme A transferases

Coenzyme A transferases (CoA-transferases) are transferase enzymes that catalyze the transfer of a coenzyme A group from an acyl-CoA donor to a carboxylic acid acceptor. Among other roles, they are responsible for transfer of CoA groups during fermentation and metabolism of ketone bodies. These enzymes are found in all three domains of life.

References

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