Citrate Lyase, CAS No.9012-83-3

CAS: 9012-83-3 Cat. No.: C1374087 分子量: >5 kDa EC番号: 232-740-7

GRADE & PURITY Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™, from Klebsiella pneumoniae ; ≥0.2 U/mg enzyme powder

Accession #

P45413,P17725

Bioactivity

≥0.2 U/mg enzyme powder

概要

Citrate lyase is an enzyme that converts citrate to oxaloacetate.

Specifications

Product Name

Citrate Lyase, CAS No.9012-83-3

同義語

Citrate oxaloacetate lyase | citrate (pro-3S)-lyasecitrase | citratase | citrate aldolase | citrate lyase | citridesmolase | citritase | citF | citE

等級

ActiBioPure™, Bioactive, High Performance, Native, EnzymoPure™

仕様と純度

Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,from Klebsiella pneumoniae ; ≥0.2 U/mg enzyme powder

生化学的・生理学的メカニズム

Represents a citryl-ACP lyase.

生物活性

≥0.2 U/mg enzyme powder

アクセス番号

P45413,P17725

CAS

9012-83-3

酵素委員会番号

EC 4.1.3.6

分子タイプ

酵素

Product Line

biological_products

保管と配送

集中

from Klebsiella pneumoniae ; ≥0.2 U/mg enzyme powder

再構成

Reconstitute in watewater to a concentration of 10 mg/ml.

保管条件

Store at -20°C,Avoid repeated freezing and thawing

入荷

Ice chest + Ice pads

安定性と保管

Store at 2-8℃ short term (1 year). Store at -20℃ long term (2 years). Upon reconstitution, it is recommended to aliquot. Avoid freeze/thaw cycle.

ユニットの定義

At pH 7.6 and 25℃, one unit converts 1.0 μmole of citrate to oxaloacetic acid per minute.

Application Protocols

The following are general, literature-based protocols for working with citrate lyase. Item-specific concentrations and activities are Not specified; consult the CoA/Spec Sheet and optimize empirically.

1. ACL activity assay (NADH-coupled):

• Prepare assay buffer: 50 mM Tris‑HCl pH 7.8, 5 mM MgCl2, 0.2 mM NADH, 0.5 mM ATP, 0.1 mM CoA.
• Add auxiliary enzyme: Malate dehydrogenase (excess, e.g., 5–10 U/mL).
• Start reaction with citrate (1 mM) and citrate lyase. Monitor A340 nm decrease at 25–30°C. Calculate rates using ε340(NADH)=6.22 mM−1 cm−1 (literature).

2. Citrate quantification (enzyme-linked method):

• In a two- or three-enzyme system, use citrate lyase to generate oxaloacetate, convert to malate via MDH, and couple to NADH oxidation for spectrophotometric detection. Construct a standard curve with known citrate standards.

3. In vitro acetyl‑CoA generation (pathway module):

• Mix citrate, ATP, CoA, Mg2+ in HEPES pH 7.5 with ACL. Couple downstream to acetyltransferase or FAS modules. Include ATP regeneration (e.g., acetate kinase/adenylate kinase) if sustained flux is required.

General tips:

• Keep all reagents on ice before mixing; pre‑warm cuvettes to assay temperature.
• Use low-binding plasticware or add 0.1 mg/mL BSA to minimize adsorption.
• Run heat‑inactivated enzyme controls to correct for background.

Biological Roles

• Central metabolic role (literature):
  • ATP‑citrate lyase (ACL) links mitochondrial citrate export to cytosolic acetyl‑CoA pools, fueling fatty acid and cholesterol biosynthesis and providing acetyl groups for protein acetylation (e.g., histones), thus influencing epigenetic regulation.
  • Bacterial citrate (pro‑3S)‑lyase participates in citrate fermentation pathways under anaerobic conditions, supporting growth on citrate by cleaving it into oxaloacetate and acetate.
• Compartmentation and regulation (literature):
  • In eukaryotes, ACL is typically cytosolic and regulated by substrate availability, phosphorylation states, and nutrient cues.
  • In bacteria, the multi‑component citrate lyase is inducible by citrate and often requires activating enzymes to generate its acyl‑enzyme intermediate.
• Systems biology relevance:
  • Flux analysis: 13C‑tracing of citrate through ACL informs lipid synthesis rates and acetyl‑CoA dynamics.
  • Crosstalk: Cytosolic acetyl‑CoA from ACL integrates carbon metabolism with acetylation signaling pathways.
• Structural/biochemical notes (literature):
  • ACL is commonly an α2β2 heterotetramer in many eukaryotes; citrate (pro‑3S)‑lyase is a multi-enzyme complex with a unique acyl‑carrier prosthetic group vital for catalysis.
• Note: The specific organism source and EC classification for this item are Not specified; consult the CoA/Spec Sheet to determine which biological context applies.

Buffer Applications

This enzyme is used in buffered aqueous systems rather than as a buffer component. The following are general, literature-based guidelines for preparing buffers for citrate lyase assays and storage. Item-specific formulation is Not specified; consult the CoA/Spec Sheet.

• Assay buffer (typical, literature):
  • 50 mM Tris‑HCl or HEPES, pH 7.4–8.0.
  • 5–10 mM MgCl2 (required for ATP‑dependent ACL activity).
  • 0.1 mM EDTA or none (avoid chelators if Mg2+ is essential).
  • 0.5–5 mM DTT or 0.5–2 mM TCEP to maintain thiols.
  • 0.1 mg/mL BSA to reduce surface adsorption (optional).
• Substrates/cofactors (as applicable):
  • Citrate (0.1–10 mM), ATP (0.5–5 mM), CoA (0.05–0.5 mM) for ACL; adjust based on Km values from literature for the specific source.
  • For bacterial citrate (pro‑3S)‑lyase, ensure the acyl‑carrier/prosthetic group is in the active (acetyl‑enzyme) state; activation may require an accessory enzyme system (literature).
• Storage buffer (general):
  • Neutral pH (7.0–8.0) with 10–50% glycerol, 100–300 mM NaCl, and reducing agent; aliquot and store at −20°C (Product Data) or colder for long term. Avoid repeated freeze‑thaw.
• Practical notes:
  • Filter-sterilize (0.2 µm) and chill buffers.
  • Validate buffer composition with small-scale pilot assays for your substrate/cofactor conditions.

Green Alternatives

Enzymatic catalysis with citrate lyase offers a greener alternative to multi‑step chemical routes for generating acetyl‑CoA equivalents or cleaving citrate under mild, aqueous conditions.

• Environmental advantages (general, literature):
  • Aqueous media at ambient temperatures reduce solvent and energy footprints.
  • High chemo-/regioselectivity lowers need for protecting groups and minimizes byproducts.
  • Compatibility with biobased feedstocks (e.g., fermentation‑derived citrate) supports circular bioprocesses.
• Comparison (general):
  • Traditional chemical approaches to activate citrate or generate acetyl equivalents often require strong dehydrating agents, chlorinating reagents, or high temperatures, producing salt waste.
  • Citrate lyase enables direct C–C bond scission or ATP‑driven acetyl‑CoA formation in one pot with benign salts as coproducts (ADP/Pi, acetate/oxaloacetate).

Simple comparison (literature-informed):

• Chemical cleavage/activation of citrate: harsh reagents, elevated temperature, organic solvents; lower selectivity; significant waste.
• Citrate lyase catalysis: water-based, near‑neutral pH, 25–37°C; high selectivity; minimal waste; enzyme sourcing and stability are the main tradeoffs.

Implementation tips:

• Couple with cofactor‑recycling modules (ATP regeneration using acetate kinase or polyphosphate kinases) to improve atom economy.
• Use immobilized enzyme formats (if available in your lab) for reuse and easier separation; validate activity retention empirically.

Pharmaceutical Uses

No therapeutic or clinical claims are made for this product. For research use only (Product Data).

In pharmaceutical and biomanufacturing research contexts (general, non-clinical):

• Enzymatic tool in metabolism studies: ACL activity informs acetyl‑CoA availability impacting lipid biosynthesis pathways during cell line development and process optimization (research setting).
• Assay development: Reference enzyme for method development of citrate, acetyl‑CoA, or oxaloacetate quantification in complex matrices (e.g., process analytics, raw material testing). Not for diagnostic use.
• Synthetic biology/biocatalysis: Component of in vitro enzymatic cascades to generate acetyl‑CoA from citrate for pathway prototyping under mild, aqueous conditions.
• Epigenetics research: Used to create defined acetyl‑CoA environments in cell‑free chromatin modification assays (literature research use).

Formulation/excipient status: No pharmacopeial monograph or excipient status is specified for this item. Any use in GMP environments requires independent qualification and documentation. Verify activity, purity, and absence of adventitious agents per your quality system. Always consult the CoA/Spec Sheet for item-specific composition and test results.

Physical Properties

• Physical state/appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Isoelectric point (pI), extinction coefficient, subunit MW: Not specified for this item; refer to CoA/Spec Sheet.
• Solubility: Enzymes are generally soluble in aqueous buffers containing salts and stabilizers (literature). Specific solubility for this lot is not specified; consult CoA.
• Stability profile: Proteins are sensitive to heat, mechanical shear, and repeated freeze–thaw (general). This item explicitly notes to avoid repeated freezing and thawing (Product Data).
• Optimal pH and temperature (literature, typical ranges):
  • ATP‑citrate lyase: pH 7.0–8.5; assay temperatures 25–37°C; requires Mg2+, ATP, and CoA (literature).
  • Bacterial citrate (pro‑3S)‑lyase: pH 7.0–8.5; 25–37°C; activity depends on intact acyl‑carrier/prosthetic group (literature).
• Cofactor requirements (literature):
  • ACL: ATP, CoA, Mg2+.
  • Bacterial enzyme: CoA derivatives/acyl-carrier forms; Mg2+ often stimulatory.
• Protein concentration measurement: UV280 nm using extinction coefficient or colorimetric protein assays (BCA/Bradford). Specific ε280 is not specified for this item.
• Note: All item-specific numeric specifications (e.g., activity units/mg, concentration, additives) are Not specified for this item; refer to CoA/Spec Sheet.

Quality and Grades

• Specified designations for this item: Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™ (Product Data).
• Interpretation (general definitions; verify specifics on CoA):
  • Bioactive: Supplied to retain enzymatic activity under recommended storage/handling.
  • Native: Isolated from a biological source rather than recombinantly produced; retains native post-translational features typical of source (general).
  • High Performance: Emphasis on performance characteristics such as activity recovery and consistency across lots; exact metrics not specified for this item.
  • ActiBioPure™ / EnzymoPure™: Proprietary purity/quality labels indicating enhanced purity and low contaminant profiles suitable for biochemical assays. Exact thresholds for residual DNA/proteins/salts, endotoxin, or specific activity are Not specified for this item; refer to CoA/Spec Sheet.
• What to check on the CoA/Spec Sheet for your application:
  • Source organism and EC classification (ACL vs citrate (pro‑3S)‑lyase).
  • Specific activity (U/mg) and assay definition.
  • Purity assessment method (SDS‑PAGE, SEC, HPLC) and aggregate level.
  • Formulation (buffer, stabilizers such as glycerol, salts, DTT) and concentration.
  • Residual nuclease/protease testing if compatibility with sensitive assays is required.
• Lot-to-lot control: For enzymology and kinetic studies, qualify a working lot and record activity under your assay conditions; enzymes can show matrix-dependent performance even at the same nominal units.

Reaction and Applications

Citrate lyase catalyzes the cleavage of citrate to yield acetyl-derived products and oxaloacetate, enabling central carbon flux analyses and biocatalytic C–C bond scission.

• Two widely used variants (literature):
  • ATP‑citrate lyase (EC 2.3.3.8): Citrate + CoA + ATP → Acetyl‑CoA + Oxaloacetate + ADP + Pi. Key for cytosolic acetyl‑CoA supply in lipid biosynthesis and histone acetylation studies.
  • Bacterial citrate (pro‑3S)‑lyase (EC 4.1.3.6): Citrate → Oxaloacetate + Acetate via a multienzyme complex with a covalent acyl‑carrier prosthetic group; no ATP consumption.
• Research applications (general):
  • In vitro acetyl‑CoA generation for fatty acid synthase or polyketide synthase assays (with ACL).
  • Coupled assays for citrate utilization, carbon flux tracing with 13C‑labeled citrate, and metabolic engineering prototyping.
  • Analytical enzymatic kits for citrate quantification in food/fermentation matrices (method development; not for diagnostic use).
  • Biocatalytic formation of oxaloacetate for downstream transamination or malate dehydrogenase-coupled readouts.
• Practical tips:
  • Confirm the EC type and required cofactors on the CoA for this item before designing assays.
  • Maintain Mg2+ for ATP‑dependent activity; pre-equilibrate buffers at working temperature.
  • For coupled assays, ensure auxiliary enzymes (e.g., malate dehydrogenase, citrate synthase) are excess to render citrate lyase rate‑limiting for accurate kinetics.
  • Minimize chelators (EDTA) and detergents; they can inhibit activity.
• Controls: Include heat‑inactivated enzyme and no‑substrate controls to establish baseline signals.

Reaction Conditions

The following are general, literature-based conditions for citrate lyase assays. Item-specific conditions (activity units, buffer composition) are Not specified; consult the CoA/Spec Sheet.

• ATP‑citrate lyase (ACL, EC 2.3.3.8):

  • Buffer: 50 mM Tris‑HCl or HEPES, pH 7.4–8.0.
  • Cofactors: 5–10 mM MgCl2, 0.05–0.5 mM CoA, 0.5–5 mM ATP.
  • Substrate: 0.1–10 mM trisodium citrate.
  • Temperature: 25–37°C; pre-equilibrate components.
  • Reducing agent: 0.5–2 mM DTT or TCEP.
  • Typical readouts: Coupled assay to measure acetyl‑CoA (via citrate synthase/DTNB) or oxaloacetate through malate dehydrogenase (NADH oxidation at 340 nm).
  • Expected ranges: Initial rates proportional to enzyme units; steady-state kinetics often follow Michaelis–Menten with Km(citrate) in 0.1–2 mM range depending on source (literature).

• Bacterial citrate (pro‑3S)‑lyase (EC 4.1.3.6):

  • Buffer: 50 mM potassium phosphate or Tris, pH 7.0–8.0.
  • Activation: Requires active acyl‑carrier prosthetic group; may need an activating enzyme and acetyl donor (literature).
  • Substrate: 0.1–10 mM citrate.
  • Temperature: 25–37°C.
  • Readouts: Oxaloacetate quantified via coupling to malate dehydrogenase (NADH → NAD+ at 340 nm) or acetate assays.

• Practical considerations:

  • Avoid EDTA and strong detergents. Maintain ionic strength (100–200 mM NaCl) to reduce nonspecific adsorption.
  • For kinetic constants, run substrate titrations with enzyme at <10% substrate consumption during initial rate windows.

Safety and Handling

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; consult the SDS for authoritative safety information.
• General laboratory precautions (proteins/enzymes):
  • Wear appropriate PPE: lab coat, gloves, and eye protection.
  • Avoid inhalation of lyophilized powders/aerosols; handle solutions gently to minimize splashes.
  • Use on ice or at 2–8°C during manipulations to preserve bioactivity.
• Storage incompatibilities: Avoid strong oxidants and proteases; do not mix with metal-chelating agents if the enzyme requires divalent cations (general). Maintain recommended buffer ionic strength and pH near neutral.
• Special risks: Enzymes may denature with heat, surfactants, organic solvents, or extreme pH. Repeated freeze–thaw cycles reduce activity—this item specifically instructs to avoid repeated freezing and thawing (Product Data).
• First-aid overview (general guidance; defer to SDS):
  • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical attention if irritation persists.
  • Inhalation: Move to fresh air; seek medical attention if symptoms occur.
  • Ingestion: Rinse mouth; seek medical advice.
• Waste disposal: Treat as laboratory biochemical waste; follow institutional and local regulations. Avoid drain disposal unless permitted by local rules and SDS guidance.

Solvent Selection

This product is a protein enzyme and is not used as an organic solvent. Solubility and performance depend on aqueous buffer systems rather than solvent choice.

• Aqueous buffer considerations (general, literature):
  • Buffer types: Tris‑HCl, HEPES, phosphate at pH 7.0–8.5 are commonly compatible.
  • Ionic strength: 50–200 mM total salt (e.g., NaCl) often stabilizes proteins and reduces nonspecific adsorption.
  • Cofactors: Include Mg2+ (1–10 mM) and, depending on EC type, ATP and CoA for ATP‑citrate lyase. Bacterial citrate lyase may require activation of its acyl‑carrier component.
  • Stabilizers: 5–50% glycerol, 0.1–1 mg/mL BSA, and 1–5 mM DTT or TCEP can enhance stability (literature). Verify compatibility with downstream assays.
• Organic co-solvents: Avoid or minimize; >5–10% organic content can denature many enzymes (literature). If unavoidable, empirically test tolerance.
• pH control: Maintain within the enzyme’s optimal window; adjust for temperature effects on buffer pKa (e.g., Tris decreases ~0.028 pH/°C; literature).
• Practical tip: Filter buffers (0.2 µm), degas if performing kinetic assays with spectrophotometry to minimize bubbles, and pre-chill solutions to preserve activity.

Storage and Reconstitution

• Storage conditions (Product Data): Store at −20°C. Avoid repeated freezing and thawing.
• Shipping: Ice chest + ice pads (Product Data). Inspect upon receipt; if thawed, promptly refrigerate and then aliquot for −20°C storage per your risk assessment.
• Reconstitution (general guidance; verify formulation on CoA):
  • If lyophilized: Briefly spin down. Reconstitute on ice with chilled buffer (e.g., 20–50 mM Tris‑HCl or HEPES, pH 7.5–8.0, 100–300 mM NaCl) containing 10–50% glycerol and 0.5–2 mM DTT/TCEP. Gently mix without vortexing.
  • If supplied in solution: Keep on ice during handling. Proceed to aliquoting immediately.
• Aliquoting: Dispense into single‑use volumes in low‑bind tubes to prevent activity loss from freeze–thaw. Label aliquots with concentration, buffer, and date.
• Working stocks: Maintain on ice during use. For short-term storage (days to weeks), −20°C with 10–50% glycerol is common. For longer-term storage, consider −80°C; confirm stability empirically for your assay.
• Freeze–thaw avoidance: Plan experiments to consume a full aliquot; do not refreeze thawed enzyme. If repeated access is unavoidable, consider adding cryoprotectants (glycerol, trehalose) and using many small aliquots.
• Light/metal sensitivity: Protect from strong light and avoid metal chelators if Mg2+ is required for activity (general).
• Documentation: Consult the CoA/Spec Sheet for item-specific concentration, buffer composition, stabilizers, and any additional storage notes. For research use only.

Structure and Identity

• Item: Citrate Lyase (enzyme preparation), SKU C1374087
• CAS: 9012-83-3
• Category: Protein/enzyme (research reagent)
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Note: enzymes are polypeptides and typically polydisperse; apparent MW depends on subunit composition and source.)
• SMILES/InChI/InChIKey: Not applicable for proteins; not specified for this item.
• Structural features (general, literature):
  • Citrate lyases exist as multi-subunit protein complexes. Two principal enzymatic classes are encountered in research:
    1. ATP-citrate lyase (ACL, EC 2.3.3.8), a cytosolic enzyme that uses ATP and CoA to convert citrate to acetyl‑CoA and oxaloacetate.
    2. Bacterial citrate (pro-3S)-lyase (EC 4.1.3.6), a multi-enzyme complex that cleaves citrate to oxaloacetate and acetate via a covalent acetyl-carrier prosthetic group (literature).
  • Typical quaternary organizations include α/β (and sometimes γ) subunits; exact subunit composition is source-dependent (literature).
• 2D/functional description: A globular protein complex with active sites that bind citrate and accessory cofactors (ATP/CoA/Mg2+ for ACL or acyl-carrier/prosthetic group for bacterial forms). Binding grooves accommodate the tricarboxylate of citrate via ionic/hydrogen-bonding interactions (literature).
• Source/EC number: Not specified for this item; refer to CoA/Spec Sheet for organism, EC classification, and specific activity.

Synthetic Utility

As a biocatalyst, citrate lyase enables selective C–C bond cleavage of citrate and ATP‑driven generation of acetyl‑CoA equivalents in aqueous media.

• Functional transformation (literature):
  • ACL: Converts citrate into acetyl‑CoA and oxaloacetate. Acetyl‑CoA is a versatile acyl donor for enzymatic acylations and starter unit for fatty acid/polyketide assembly lines.
  • Bacterial citrate lyase: Cleaves citrate to acetate and oxaloacetate, supplying C2/C4 building blocks under mild conditions.
• Retrosynthetic value:
  • Facilitates modular in vitro pathways where acetyl‑CoA supply is rate‑limiting (e.g., coupling with acetyltransferases, thiolases, or fatty acid synthase).
  • Generates oxaloacetate for subsequent enzymatic conversions (e.g., transamination to aspartate, reduction to malate).
• Coupled systems:
  • Combine with malate dehydrogenase and lactate dehydrogenase for NADH-linked readouts.
  • Use acetate kinase/adenylate kinase/pyrophosphate systems for ATP recycling; CoA can be conserved via thiolase cycles (literature).
• Practical implementation:
  • Maintain cofactor balance (ATP/Mg2+/CoA) and remove inhibitory inorganic phosphate where necessary.
  • Immobilization or encapsulation can improve operational stability; empirically determine turnover numbers for your matrix.
• Note: Specific activity, optimal conditions, and tolerance to salts/solvents for this item are Not specified; refer to CoA/Spec Sheet and perform small-scale optimization.

Target Specificity

This product is an enzyme, not an affinity reagent. Target/epitope specificity fields (antigen name, clone, isotype, species reactivity) are not applicable.

• Item-specific binding targets: Not applicable.
• If you require an antibody against citrate lyase or a specific organism’s isoform, please refer to antibody products; this SKU C1374087 is an enzymatic preparation intended for catalytic applications.