Enzymes II

Enzymes II

1. Inhibition

   • Irreversible
     • Bind very tightly or covalently modify the enzyme.
     • Modify AA side chains
       • Irreversible reactions
     • Sometimes very tight binding
       • No chemical reaction
     • Allopurinol
       • Inhibits Xanthine Oxidase
         • Required for purine breakdown
         • Irreversibly binds to active site
       • Treats gout
     • Organophosphorous pesticides/nerve gas (DIFP)
       • Inhibits acetylcholinesterase
         • Binds active site
           • AAs
             • Aspartate, Histidine, Serine, and Glycine
       • Inhibits serine proteases
     • Aspirin
       • Inhibits prostaglandin/thromboxane synthesis
         • Anti-inflammatory
   • Reversible
     • Reversible by dialysis or dilution
     • Competitive
       • Alters apparent Km
         • Increase Km
         • Vmax unchanged
       • Amethopterin
         • Competitive inhibitor
           • Dihydrofolate reducatse
           • Binds tightly to active site
             • Inhibits purine synthesis
               • Used for chemotherapy
       • Ethanol
         • Competitive inhibitor
           • Alcohol dehydrogenase
             • Treat methanol/ethylene glycol poisoning
     • Non-competitive
       • Doesn't bind to active site
       • Vmax decreased
       • Km is unchanged
     • Anti-inflammatories
       • Prevent prostaglandin/thromboxane synthesis
       • Ibuprofen
       • Acetaminophen

2. Physiological Conditions

   • pH
     • Protonated or deprotonated AAs are often required for an active enzyme i.e., Chymotrypsin
     • Rxn requires deprotonated AA
       • At pKa, 0.5 of side chains deprotonated
         • Rate = 0.5Vmax
     • Rxn requires protonated AA
       • At pKa, 0.5 of chains are protonated
         • Rate = 0.5Vmax
     • Dependency
       • Produces bell shaped curve around pKa
   • Temperature
     • Rates increase with increase in temp
       • ~2x/10 C
     • At higher temps, rate decreases drastically
       • Often irreversible
       • Denaturing of enzyme
       • Mutations lead to less stability earlier
         • Glucose 6-P Dehydrogenase
           • This enzyme is important to maintain RBC membrane. The product NADPH is a substrate for glutathione reductase.
           • Glucose 6-P + NADP --> 6-phosphogluconate + NADPH NADPH + G-SS-G --> GSH + NADP
           • Lack of reduced GSH leads to formation of peroxides which react with unsaturated fatty acids in membrane of cells --> anemia
           • This mutation is especially important under conditions of oxidative stress or prolonged fever.
           • Anemia

3. Kinetics

   • Michaelis-Menton Equation
     • v = Vmax / 1+Km/S
       • At S = Km
         • V = Vmax / 2
       • Km is affinity
         • Lower Km, higher affinity
           • Hexokinase Km < Glucokinase Km
             • Hexokinase active at lower [substrate]
               • Peripheral tissue
         • Substrate concentration for 0.5 Vmax
         • Acetaldehyde Dehydrogenase
           • Mitochondria have ALDH with low KM
             • Remove from blood faster
             • Asians missing this form
               • Higher sensitivity to alcohol
               • Only have high Km form
                 • Acetaldehyde stays in blood longer
       • Vmax is rate
         • Saturating substrate
         • 5'-phosphoribosyl-pyrophosphate synthase
           • Precursor of purine nucleotide
           • Mutation will increase Vmax
             • Vmax will increase to 3x the normal rate
             • Excess purine synthesis
               • Leads to excess uric acid
                 • Gout