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.
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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 __ketoglutarate, feeding the TCA cycle.
Low glucose / fasting Pyruvate carboxylase activity rises; __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 CO_ for energy generation.
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4. Physiological role _ metabolic and clinical implications
Energy metabolism
Generates high_yielding NADH that feeds the electron transport chain.
CO_ 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.
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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.
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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.
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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 His_ 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 Ni
