Biochemistry: Enzyme Inhibitors

Inhibitors

• Inhibitors bind more tightly to an enzyme than substrates or products.
• Many pharmacological examples exist, such as penicillin and allopurinol.
• Allopurinol is an example of a transition state analog - it inhibits by binding to the active site of the enzyme that makes uric acid.

Reversible Inhibition

• Competitive Inhibition: The inhibitor binds to the active site of an enzyme.
• Noncompetitive Inhibition: The inhibitor binds to a site on the enzyme other than the active site (allosteric site).
  • It can bind before or after a substrate binds.
  • Pure noncompetitive inhibition occurs when the inhibitor binds to a site different from the substrate. The enzyme may have one or more substrates.
  • Mixed noncompetitive inhibition occurs when the inhibitor binds outside of the substrate-binding sites.
• Uncompetitive Inhibition: The inhibitor only binds to the enzyme-substrate complex, but not to the enzyme alone.
  • This only happens in multi-substrate reactions.

Lineweaver-Burk Plots

• Using the reciprocal of both velocity and substrate concentration, a hyperbolic curve can be transformed into a straight line.
• This transformation makes it easier to compare different conditions and determine the type of inhibition.

Competitive Inhibition

• Vmax remains unaffected.
  • The definition of Vmax is the maximum velocity at which the enzyme works when there is an unlimited amount of substrate.
  • The inhibitor has no effect if there is an unlimited amount of substrate.
• Km is affected.
  • The apparent Km (Km,app) is increased with the inhibitor.
  • This means that the enzyme's affinity for substrate is decreased.
• Example: Hemoglobin binding oxygen.

Cooperativity

• Activators:
  • Increase the affinity of the enzyme for the substrate.
  • Tend to bind more tightly to the R state.
• Inhibitors:
  • Decrease the affinity of the enzyme for the substrate.
  • Tend to bind more tightly to the T state.
• Activators shift the sigmoid-shaped substrate saturation curve to the left.
• Inhibitors shift the curve to the right.
• Example: Phosphofructokinase-1 (PFK-1) in glycolysis.

Enzymatic Regulation

• Regulation by Substrate Availability: Changes in the concentration of the substrate can change the reaction velocity.
  • This is a fast response, occurring over seconds.
• Product Inhibition: Accumulation of the product can inhibit the reaction, decreasing the apparent Vmax and/or Km.
  • This is a fast response, occurring over seconds.
• Allosteric Control: Binding of an effector molecule to an allosteric site can change the enzyme's activity, altering the Vmax and/or Km.
  • This is also a fast response, occurring over seconds.
• Covalent Modification: The covalent modification of an enzyme, such as phosphorylation, can alter its activity.
  • This change occurs over seconds to minutes, or even days.
• Induction or Repression: Changes in the amount of enzyme protein can be caused by hormones or metabolites, which can occur over minutes, hours, or days.

PFK-1 Regulation

• PFK-1 is the rate-limiting enzyme in glycolysis.
• Activators: AMP and fructose-2,6-bisphosphate (F-2,6-BP).
• Inhibitors: Citrate and ATP.

F-2,6-BP

• Glucose is converted to fructose-6-phosphate (F-6-P) which is then converted to fructose-2,6-bisphosphate (F-2,6-BP) by the enzyme PFK-2.
• F-2,6-BP is then converted back to F-6-P by the enzyme F-2,6-bisPase.
• PFK-1 is activated by F-2,6-BP.
• Levels of F-2,6-BP determine how much PFK-1 activity occurs.

PFK-2 & F-2,6-BPase

• PFK-2 and F2,6-BPase can be phosphorylated and dephosphorylated.
  • In heart and skeletal muscle, the enzymes are regulated by the concentration of the substrate.
• In the liver:
  • PFK-2 is active when it is dephosphorylated and inactive when it is phosphorylated.
  • F2,6-BPase is inactive when it is dephosphorylated and active when it is phosphorylated.
• The liver regulates glycolysis and gluconeogenesis based on these changes.
• Adipose tissue regulates glycolysis based on changes in these enzymes.

Phosphofructokinase-1

• Regulated allosterically by AMP & F-2,6-BP.
• There is no phosphorylation site in skeletal muscle PFK-2.
  • It is regulated by the availability of the substrate.
• There is a cAMP-dependent protein kinase (PKA) phosphorylation site in liver PFK-2.

Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)

• Catalyzes a reversible reaction found in both glycolysis and gluconeogenesis.
• Produces the high-energy molecule 1,3-bisphosphoglycerate (1,3-BPG) and reduces NAD+ to NADH.
• Inactivated by reaction with iodoacetate, iodoacetamide, arsenite, or mercury ions.
• Arsenate (AsO43-) can substitute for phosphate (PO43-).
  • This leads to a futile cycle, which is a potential treatment for cancer cells that rely heavily on glycolysis.

Fructose Metabolism

• Fructose bypasses the rate-limiting step of glycolysis, PFK-1, making fructose metabolism less regulated.
  • High fructose corn syrup consumption (about 50% fructose) is associated with fatty liver and hyperglycemia.
• Fructokinase deficiency leads to fructosuria.
• Aldolase B deficiency leads to hereditary fructose intolerance.

Fructokinase Deficiency

• Fructosuria: A rare but benign condition (autosomal recessive).
  • Individuals with this condition have high blood fructose levels.

Aldolase B Deficiency

• Hereditary fructose intolerance: An autosomal recessive condition.
  • Aldolase B is the rate-limiting enzyme in fructose catabolism.
  • Individuals with this deficiency accumulate F-1-P in their livers.
    • This depletes liver phosphate levels, which are trapped as F-1-P.
  • Symptoms are severe: diarrhea, vomiting, failure to thrive, liver and kidney damage.
  • Untreated, it can lead to death.
  • Dietary restrictions are necessary: Avoid fruits, fruit juices, maple syrup, etc.

PDH Complex

• PDH - Pyruvate dehydrogenase complex.
• Composition:
  • 5 cofactors: Thiamine pyrophosphate (B1), lipoic acid, CoA (B5, pantothenic acid), FAD (B2, riboflavin), NAD+ (B3, niacin).
• Role: Catalyzes the conversion of pyruvate to acetyl-CoA - the link between glycolysis and the TCA cycle.

PDH Complex Regulation

• Substrate activation and product inhibition:
  • Activators: Pyruvate, CoA, and NAD+.
  • Inhibitors: Acetyl-CoA and NADH.
• Covalent modification:
  • Phosphorylation and dephosphorylation of the complex by hormones.
    • Insulin is an activator of dephosphorylation, which leads to activation of the complex, in the liver.
• Allosteric regulation:
  • The complex can be activated or inhibited by allosteric effectors.

PDH Deficiency

• Symptoms: Increased levels of pyruvate, lactate, and alanine.
  • Chronic lactic acidosis.
  • Severe neurological defects that could eventually lead to death.

TCA Cycle Regulation

• 3 Steps:
  • Citrate synthase (inhibited by ATP, NADH, and acetyl-CoA).
  • Isocitrate dehydrogenase (activated by ADP and NAD+, inhibited by ATP and NADH).
  • α-ketoglutarate dehydrogenase (inhibited by succinyl CoA, NADH, and ATP).
• Location: Inside the mitochondrial matrix, except succinate dehydrogenase, which is located in the inner mitochondrial membrane.

TCA Cycle - Energy Production and Investment

• Energy Investment:
  • 1 ATP equivalent (GTP) is produced per turn of the cycle.
• Energy Production:
  • 3 NADH molecules are produced per turn.
  • 1 FADH2 molecule is produced per turn.
  • These reduced cofactors provide the electrons to the ETC for ATP generation.

Catapleurotic/Anapleurotic Reactions

• Catapleurotic reactions: Reactions that remove intermediates from the TCA cycle.
• Anapleurotic reactions: Reactions that replenish intermediates in the TCA cycle.
• Pyruvate carboxylase deficiency: A rare genetic condition that affects the anapleurotic pathway.
  • This can disrupt the proper functioning of the TCA cycle.

Pentose Phosphate Pathway (PPP)

• Two Phases:
  • Oxidative phase: This phase generates NADPH and the precursor for nucleotide biosynthesis (ribose 5-phosphate).
  • Non-oxidative phase: This phase interconverts pentose phosphates (5-carbon sugars) to other sugars like glucose-6-phosphate.
• Regulation:
  • The PPP responds to different metabolic needs.
    • Increased NADPH production: When cells need to reduce oxidative stress.
    • Increased nucleotide biosynthesis: When cells need to synthesize nucleic acids.

Explore the fascinating world of enzyme inhibitors in this quiz. Learn about different types of inhibition, including competitive, noncompetitive, and uncompetitive inhibition. Test your knowledge on pharmacological examples like allopurinol and how inhibitors interact with enzymes.