Acceptor oxidoreductase; Phenylacetyl CoA serves as a substrate for Phenylacetyl-CoA:acceptor oxidoreductase (EC 1.17.99.-). This enzyme catalyzes the four-electron oxidation of phenylacetyl-CoA to phenylglyoxylate and releases CoASH. The apparent Michaelis constant (Km) of the enzyme for phenylacetyl-CoA is 20 μM.[1]

- The purified phenylacetyl-CoA:acceptor oxidoreductase catalyzes the reaction: Phenylacetyl-CoA + 2 quinone(ox) + 2 H₂O → Phenylglyoxylate + 2 quinone(red) + CoASH. The oxygen atom introduced into the product is derived from water.[1]
- The enzyme exhibits relative substrate specificity. Among various CoA esters tested (0.1 mM), only phenylacetyl-CoA was oxidized. No reaction was observed with phenylacetate, acetyl-CoA, propionyl-CoA, succinyl-CoA, crotonyl-CoA, benzoyl-CoA, or 3-hydroxybenzoyl-CoA.[1]
- The enzyme accepts various electron acceptors. At 0.3 mM, duroquinone showed the highest relative activity (150% compared to DCPIP at 100%), followed by menadione (60%). The estimated catalytic turnover number with DCPIP as electron acceptor was 1-2 s⁻¹.[1]
- HPLC analysis revealed that the intermediate phenylglyoxylyl-CoA was transiently formed (approximately 0.03 mM from 0.1 mM phenylacetyl-CoA). Free mandelate or mandelyl-CoA was not detected. All enzyme preparations hydrolyzed phenylglyoxylyl-CoA but not phenylacetyl-CoA.[1]

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Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine. In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (benzoylformate). The enzyme that catalyzes the four-electron oxidation of phenylacetyl-CoA has been purified from this bacterium and studied. The enzyme preparation catalyzes the reaction phenylacetyl-CoA + 2 quinone + 2 H2O --> phenylglyoxylate + 2 quinone H2 + CoASH. Phenylacetyl-CoA:acceptor oxidoreductase is a membrane-bound molybdenum-iron-sulfur protein. The purest preparations contained three subunits of 93, 27, and 26 kDa. Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water. The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa. Phenylglyoxylyl-CoA, but not mandelyl-CoA, was observed as a free intermediate. All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl-CoA but not from phenylacetyl-CoA. The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen. This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha-oxidation of a CoA-activated carboxylic acid without utilizing molecular oxygen [1].

In vivo studies demonstrate that phenylacetic acid (the precursor of Phenylacetyl CoA) suppresses gluconeogenesis. In both normal and streptozocin-induced diabetic rats, intravenous administration of phenylpropionic acid (a phenylacetic acid analog) significantly decreased blood glucose levels (normal rats: 110 ± 12 to 66 ± 11 mg/dL; diabetic rats: 295 ± 14 to 225 ± 12 mg/dL). This glucose-lowering effect is attributed to the inhibition of pyruvate carboxylase by Phenylacetyl CoA in the liver, thereby reducing de novo glucose synthesis. In clinical applications, phenylacetic acid is converted to Phenylacetyl CoA, which subsequently conjugates with glutamine to form phenylacetylglutamine, serving as an ammonia scavenger for patients with urea cycle disorders.

Spectrophotometric assay for phenylacetyl-CoA:acceptor oxidoreductase activity: The reaction was carried out under anoxic conditions at 30°C in a total volume of 0.3 mL. The assay mixture contained 50 mM potassium phosphate buffer (pH 7.5), 0.25 mM DCPIP as artificial electron acceptor, and 20 μL of enzyme solution. The reaction was started by adding 0.1 mM phenylacetyl-CoA. The reduction of DCPIP was monitored spectrophotometrically at 546 nm (extinction coefficient ε₅₄₆ = 13,800 M⁻¹·cm⁻¹). A stoichiometry of 2 mol DCPIP reduced per mol of phenylacetyl-CoA oxidized was used for calculations. The pH optimum was determined using 50 mM phosphate buffers ranging from pH 6.0 to 8.0. The Km value was determined by varying the phenylacetyl-CoA concentration (5-200 μM) at a fixed DCPIP concentration of 0.25 mM.[1]
- Substrate specificity test: Various CoA esters (0.1 mM) were tested, including acetyl-CoA, propionyl-CoA, succinyl-CoA, crotonyl-CoA, benzoyl-CoA, and 3-hydroxybenzoyl-CoA.[1]
- Electron acceptor specificity test: In the presence of 0.1 mM phenylacetyl-CoA, the following electron acceptors (0.3 mM) were tested: DCPIP, duroquinone, and menadione. Activity was measured by determining the formation of the product phenylglyoxylate by HPLC.[1]
- Reversible cyanide inactivation experiment: The enzyme solution was preincubated with varying concentrations of cyanide (5-20 μM) at pH 7.5 for different time periods. Following preincubation, 20 μL of the enzyme solution was injected into 0.3 mL of assay mixture to start the reaction, and enzyme activity was monitored spectrophotometrically. Control enzyme was preincubated in the absence of cyanide. The results showed a time- and cyanide concentration-dependent decrease in initial activity, but activity gradually recovered during the assay, indicating reversible inactivation.[1]

In cellular assays, Phenylacetyl CoA is used to study gluconeogenesis regulation. A typical protocol involves isolating rat hepatocytes and suspending them in Krebs-Henseleit buffer. Cells are incubated with phenylacetic acid (4 mM), which is naturally converted to Phenylacetyl CoA intracellularly. Gluconeogenic activity is assessed by measuring the incorporation of [¹⁴C]-bicarbonate into glucose or the rate of pyruvate carboxylation. Studies show that phenylacetic acid (mediated by Phenylacetyl CoA) inhibits gluconeogenesis from 10 mM lactate/1 mM pyruvate by 39% (P < 0.05).

In animal models, Phenylacetyl CoA is typically studied indirectly by administering its precursor, phenylacetic acid or phenylbutyrate. A typical protocol (rat gluconeogenesis suppression model): Male Sprague-Dawley rats (200-250 g) are fasted overnight to deplete liver glycogen. Phenylpropionic acid (a phenylacetic acid analog) is administered via jugular vein catheter as a 20 mg bolus followed by continuous infusion at 1 mg/min. Blood samples are collected from the tail vein at various time points, and glucose levels are measured by glucose oxidase method. At the endpoint, animals are euthanized, and liver tissues are collected for glycogen content and pyruvate carboxylase activity assays.

Direct pharmacokinetic parameters for Phenylacetyl CoA itself as an intracellular transient intermediate are limited in the literature. However, pharmacokinetic data for its precursor, phenylacetic acid, have been reported: the time to reach maximum plasma concentration (Tmax) is approximately 3.7 hours, maximum plasma concentration (Cmax) is 205.8 μM, plasma protein binding is approximately 51%, and elimination half-life (t½) is 1.15 hours. Phenylacetyl CoA is generated in vivo from phenylacetic acid via acyl-CoA synthetase and is rapidly metabolized, primarily conjugating with glutamine to form phenylacetylglutamine for renal excretion.

Phenylacetyl CoA is non-toxic at normal physiological concentrations as an endogenous metabolite, but its precursor phenylacetic acid exhibits neurotoxicity at high concentrations. Studies suggest that the neurotoxicity of phenylacetate may be mediated by its metabolite Phenylacetyl CoA: Phenylacetyl CoA reduces acetylcholine synthesis by inhibiting choline acetyltransferase (ChAT, Ki = 3.1 × 10⁻⁷ M), which may represent a mechanism underlying the neurological damage in phenylketonuria (PKU) patients. In clinical use, when phenylacetic acid is used as an ammonia scavenger, blood concentrations must be monitored to avoid toxicity. When used as a chemical reagent, standard laboratory practices should be followed.

[1].Phenylacetyl-CoA:acceptor oxidoreductase, a membrane-bound molybdenum-iron-sulfur enzyme involved in anaerobic metabolism of phenylalanine in the denitrifying bacterium Thauera aromatica. Eur J Biochem. 1999;262(2):507-515.

- Background and metabolic pathway: In the denitrifying bacterium Thauera aromatica, anaerobic metabolism of phenylalanine proceeds via a peripheral pathway and a central pathway. In the peripheral pathway, phenylacetyl-CoA is converted to phenylglyoxylate, which is further oxidized to benzoyl-CoA. Phenylacetyl-CoA:acceptor oxidoreductase catalyzes the four-electron oxidation, the key step in the conversion of phenylacetyl-CoA to phenylglyoxylate.[1]
- Induction of the enzyme: The enzyme is induced when cells are grown anaerobically on phenylalanine or phenylacetate. Lower induction is observed when cells are grown on phenylglyoxylate or benzoate, and weak activity is also detected when cells are grown aerobically on phenylacetate.[1]
- Summary of enzyme properties: The enzyme is a membrane-bound molybdenum-iron-sulfur protein with an estimated native molecular mass of approximately 280 kDa, composed of three subunits (93 kDa, 27 kDa, and 26 kDa). It contains approximately 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of 280 kDa enzyme. The enzyme exhibits optimal activity at pH 7.0. It is reversibly inactivated by low concentrations of cyanide but is remarkably stable with respect to oxygen.[1]

C29H42LIN7O17P3S

892.607768535614

892.173

108321-26-2

7532-39-0

6419738

Typically exists as solid at room temperature

2.169

9

22

22

58

1530

0

S(C(CC1C=CC=CC=1)=O)CCNC(CCNC(C(C(C)(C)COP(=O)(O)OP(=O)(O)OCC1C(C(C(N2C=NC3C(N)=NC=NC2=3)O1)O)OP(=O)(O)O)O)=O)=O.[Li]

PQZFFEIEACTQRR-UHFFFAOYSA-N

InChI=1S/C29H42N7O17P3S.Li/c1-29(2,24(40)27(41)32-9-8-19(37)31-10-11-57-20(38)12-17-6-4-3-5-7-17)14-50-56(47,48)53-55(45,46)49-13-18-23(52-54(42,43)44)22(39)28(51-18)36-16-35-21-25(30)33-15-34-26(21)36;/h3-7,15-16,18,22-24,28,39-40H,8-14H2,1-2H3,(H,31,37)(H,32,41)(H,45,46)(H,47,48)(H2,30,33,34)(H2,42,43,44);

phenyl acetyl Coa; 108321-26-2; Phenylacetyl coenzyme A lithium salt; S00346a; Phenylacetyl CoA-lithium; Phenylacetyl-CoA Li salt; DTXSID80423551; Phenylacetyl coenzyme A lithium salt, ~95%

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)