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  Oxandrolone Wikipedia
  
  Aldolase (also known as fructose bisphosphate aldolase) is an enzyme that
  catalyzes the reversible cleavage of fructose‑bisphosphate into two three‑carbon sugars,
  dihydroxyacetone phosphate and glyceraldehyde‑3‑phosphate.
  
  It plays a central role in both glycolysis (the breakdown of
  glucose) and gluconeogenesis (the synthesis of glucose).
  The enzyme is highly conserved across all domains of life and
  exists in several isoforms that differ in tissue distribution and
  regulatory properties.
  
  
  
  ---
  
  
  
  
  1. Classification and Isoforms
  
  
  
  
  Isoform Gene(s) Tissue Distribution Key Regulatory Features
  
  
  GAPDH‑C (cytosolic) GAPDHC Widely expressed, high in liver, heart, skeletal muscle Activated
  by NAD⁺; inhibited by ADP, ATP, and various post‑translational modifications (acetylation, phosphorylation)
  
  
  GAPDH‑A (muscle‑specific) GAPDA Predominantly in cardiac
  & skeletal muscle More resistant to oxidative stress; distinct phosphorylation pattern at Thr-25
  
  
  GAPDH‑B (mitochondrial precursor) GAPDB Imported into
  mitochondria, functions in mitochondrial matrix Requires cleavage of N‑terminal targeting sequence; NAD⁺‑dependent
  
  
  > Note: These subtypes differ mainly by N‑terminal extensions that determine cellular localization and subtle catalytic properties.
  Their kinetic parameters are comparable: \(K_m\) ≈ 0.1–0.2 mM for
  NAD⁺, \(V_max\) varies with enzyme concentration.
  
  
  
  
  3. Catalytic Mechanism
  
  
  The reaction catalyzed by GAPDH proceeds through a two‑step Ping‑Pong mechanism involving covalent intermediates:
  
  
  
  
  Step Substrate / Product Key Residues Intermediate
  
  
  1 1,3‑BPG + NAD⁺ → Glyceraldehyde‑3‑phosphate (GAP) + NADH + H⁺ Lysine 229 (covalent binding),
  His 195, Glu 232, Ser 147 (for proton transfer) Enzyme–O‑phospho‑glyceraldehyde intermediate
  
  
  2 GAP + Pi → 1,3‑BPG Same residues Regenerated enzyme
  
  
  
  
  
  
  Lys229 forms a Schiff base with the aldehyde of BPG.
  
  
  
  His195 acts as a general acid/base.
  
  
  Glu232 stabilizes the transition state.
  
  
  
  
  
  
  
  4. Metabolic context – glycolysis / gluconeogenesis
  
  
  
  Condition Role of E1 (KHGDH)
  
  
  High glucose Increases flux through glycolysis → pyruvate → acetyl‑CoA; KHGDH contributes to the production of α‑ketoglutarate, feeding the
  TCA cycle.
  
  
  Low glucose / fasting Pyruvate carboxylase activity rises; α‑ketoglutarate is needed for anaplerotic reactions and gluconeogenesis.
  The enzyme helps replenish TCA intermediates.
  
  
  Amino acid catabolism (lysine, threonine) Supplies KHGDH with substrates, enhancing production of NADH and CO₂ for energy generation.
  
  
  ---
  
  
  
  
  4. Physiological role – metabolic and clinical implications
  
  
  
  
  
  Energy metabolism
  
  
  Generates high‑yielding NADH that feeds the electron transport chain.
  
  CO₂ produced is removed via the respiratory system; the enzyme’s activity
  helps maintain acid–base balance by influencing bicarbonate production from TCA intermediates.
  
  
  
  
  
  
  Amino‑acid catabolism and nitrogen handling
  
  
  Catabolizes lysine, threonine, and other amino
  acids, thereby contributing to urea cycle precursors (ammonia).
  
  
  Provides substrates for gluconeogenesis when glucose is
  scarce.
  
  
  
  
  
  Clinical relevance
  
  
  
  - Deficiency or mutation: Rare metabolic disorders may
  arise from defective NADH‑producing enzymes in the TCA cycle, leading to energy deficits, hypoglycemia, and neurological symptoms.
  
  
  - Inborn errors of amino‑acid metabolism: Disorders such as
  hyperlysinemia involve impaired lysine catabolism, causing elevated plasma lysine levels,
  developmental delays, and seizures.
  
  - Therapeutic interventions: Supplementation with NAD⁺ precursors (e.g., nicotinamide riboside) or cofactor vitamins (B1, B2,
  B3, B5) can help restore metabolic fluxes.
  
  
  
  ---
  
  
  
  
  Summary of Key Points
  
  
  
  Aspect Details
  
  
  Enzyme Typically a dehydrogenase (often NAD⁺‑dependent).
  
  
  
  Cofactor NAD⁺/NADH, sometimes NADP⁺/NADPH.
  
  
  Reaction Oxidation of substrate + reduction of cofactor →
  product + reduced cofactor.
  
  
  Energy Yield Directly contributes to the reducing power for ATP synthesis (via oxidative phosphorylation).
  
  
  
  Disease Links Mutations or dysfunctions in the enzyme/cofactor system cause metabolic disorders, mitochondrial
  diseases, and can affect overall energy homeostasis.
  
  
  
  ---
  
  
  
  
  Quick Reference Table
  
  
  
  Step Reaction Type Co‑factor Involved Energy Impact
  
  
  1 Oxidation (substrate loses electrons) NAD⁺/NADP⁺ → NADH/NADPH Generates
  reducing equivalents for ATP
  
  
  2 Reduction (acceptor gains electrons) NAD⁻H + H⁺ → NAD⁺
  Restores co‑factor to oxidized form
  
  
  
  
  
  
  If the step is an oxidation, it’s energetically favorable because it produces
  usable energy.
  
  
  If it’s a reduction, it consumes energy and must be driven by other processes (e.g., electron transport).
  
  
  
  
  Key Takeaway: The direction of the reaction—whether electrons are being transferred from substrate to
  co‑factor or vice versa—determines whether the step
  generates or uses energy.
  
  
  
  
  
  3. How the Enzyme Uses the Energy
  
  
  The enzyme Cytosolic NAD⁺ Reductase (NADH‑dependent) harnesses the energy stored in the electron transfer chain through a cofactor cycling mechanism:
  
  
  
  
  
  
  Substrate Binding
  
  
  - The enzyme binds a substrate that will donate electrons to NAD⁺,
  forming NADH.
  
  
  
  Electron Transfer & Cofactor Regeneration
  
  
  - Electrons from the reduced substrate are transferred to
  NAD⁺ via an intermediate cofactor (e.g., FAD or a metal cluster).
  
  - This transfer creates an oxidized form of the cofactor and reduces
  NAD⁺ to NADH.
  - The oxidized cofactor then re‑reduces another
  NAD⁺ molecule, regenerating its reduced state.
  
  
  
  
  
  Catalytic Cycle Completion
  
  
  - The enzyme returns to its initial state ready for a new substrate.
  
  
  
  
  Overall Reaction
  
  
  - Net: NAD⁺ + Substrate (reduced) → NADH + Substrate (oxidized).
  
  
  The catalytic cycle ensures efficient electron transfer while maintaining
  the integrity of the cofactor, which acts as an intermediate carrier between the enzyme
  and NAD⁺/NADH.
  
  
  
  ---
  
  
  
  
  5. Applications
  
  
  
  
  Metabolic Engineering
  
  
  - By introducing this enzyme into organisms that produce valuable metabolites (e.g., biofuels), we can redirect reducing power to produce more reduced compounds
  or regenerate NAD⁺ for continuous operation.
  
  
  
  Synthetic Biology Circuits
  
  
  - The reaction can serve as a controllable switch in metabolic pathways, where the presence of
  reduced substrates drives NADH formation, potentially influencing
  downstream reactions (e.g., fermentation).
  
  
  
  Redox Biocatalysis
  
  
  - Coupled with other enzymes that consume NADH, this reaction can power cascades for selective reductions or oxidation processes in vitro.
  
  
  
  
  Industrial Enzyme Production
  
  
  - The engineered enzyme may be used to produce NADH in bulk for pharmaceutical synthesis (e.g., stereoselective reductions) without the need for expensive cofactors.
  
  
  
  
  5. Experimental Validation Plan
  
  
  
  5.1. Gene Construction and Expression
  
  
  
  Synthesize the codon‑optimized gene encoding the engineered protein.
  
  
  Clone into an expression vector with a strong promoter (e.g.,
  T7), include a His₆ tag for purification.
  
  
  Transform into E. coli BL21(DE3) or a suitable host.
  
  
  
  
  5.2. Protein Purification
  
  
  
  Induce expression, harvest cells, lyse by sonication or French press.
  
  
  
  
  Clarify lysate by centrifugation; apply to Ni²⁺‑NTA affinity column.
  
  
  Elute with imidazole gradient; further purify via size‑exclusion chromatography
  (SEC) if needed.
  
  
  
  
  5.3. Enzyme Activity Assays
  
  
  
  Substrate preparation: Synthesize or obtain the engineered substrate (e.g.,
  a fluorogenic ester containing an unnatural protecting group).
  
  
  
  Reaction conditions: Standard buffer (pH 7.0–8.0), temperature
  (25–37 °C). Include controls without enzyme.
  
  
  
  Detection methods:
  
  
  - Fluorescence spectroscopy if the substrate releases a fluorescent moiety upon cleavage.
  
  - High‑performance liquid chromatography (HPLC) to monitor disappearance of
  substrate and appearance of products.
  - Mass spectrometry (MS) for product identification, confirming removal of the protecting group.
  
  
  
  
  
  Kinetic analysis: Measure initial rates at varying substrate concentrations; fit data to Michaelis–Menten equation to obtain k_cat
  and K_M.
  
  
  
  
  Expected Outcomes
  
  
  
  
  Catalytic activity will be evident if the engineered enzyme reduces the concentration of the protected substrate and generates
  products lacking the protecting group.
  
  
  The magnitude of catalytic efficiency (k_cat/K_M) will indicate how well the active site accommodates the new chemistry.
  
  
  
  If activity is low, iterative rounds of design (e.g., adding
  additional mutations, optimizing ligand orientation) can be
  pursued.
  
  
  
  
  
  
  
  4. Summary
  
  
  
  
  Design a protein that has an internal cavity or pocket
  capable of binding the target molecule and presenting it to catalytic
  residues; use computational modeling for precise placement.
  
  
  
  Engineer catalytic sites by introducing amino acids with suitable side‑chain chemistries (acid/base, metal ligands) into the
  active site, guided by known mechanisms or directed evolution.
  
  
  Validate the design experimentally, starting with in silico docking and energy calculations, then proceed to protein expression, purification,
  structural confirmation, and kinetic assays.
  
  
  
  This approach integrates rational computational design with iterative
  experimental testing, providing a clear pathway for creating novel proteins that bind
  specific molecules and catalyze desired chemical reactions.