Enzyme Activity and Bioenergetics

Enzyme Activity and Bioenergetics

1. Kinetics

   • Zeroth order: Rate = k[A]^0
   • First order: Rate = k[A]^1
   • Second order: Rate = k[A]^2
   • Rate determining step has highest transition state energy

2. General Properties of Enzymes

   • Accelerate reaction rates, sometimes as much as 10^5-10^14
   • DO NOT CHANGE K VALUE!!
     • Lower activation energy
   • Have a capacity for regulation
   • Reaction specificity
     • Produce products in very high yields
     • Specificity determined by structure
   • Classified according to rxn type
     • Lyases
       • Cleaves C--C, C--O, C--N or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds
     • Isomerases
       • Transfer of groups within molecules to yield isomeric forms
     • Ligases
       • Formation of C--C, C--s, C--O, and C--N bonds by condensation reactions coupled to cleavage of ATP
     • Oxidoreductases
       • Transfer electrons (hydride ions or H atoms)
     • Transferases
       • Group transfer reactions
     • Hydrolases
       • Hydrolysis reactions, transfers functional groups to water
   • Must be under mild conditions
   • Require cofactors often
     • Inorganics are essential ions or cofactors, organics are coenzymes
     • Apoenzyme is inactive form w/out cofactor, holoenzyme (is whole) is active with cofactor

3. Mechanisms of Enzymes

   • Binding Energy
     • Ask Woolridge
     • Enzymes preferentially bind to the transition state
   • General Acid-Base Catalysis
     • Proton transfers are most common (10^2-10^5 rate enhancement)
     • AA residues can serve as catalysts
     • "Specific" involves H+ or OH- that diffuses into the catalytic center
     • "General" involves acids and bases other than H+ and OH-
   • Covalent Catalysis
     • Involves transient covalent bonds forming between enzyme and substrate
     • AAs that can do acid-base can do this as well
   • Metal Ion Catalysis
     • Mediate redox rxns
     • Stabilize charges
     • Ionize water

4. Enzyme Kinematics

   • E + S -><- ES --> E + P
     • ES is Michaelis complex of enzyme and substrate
   • v = Vmax[S]/Km + [S]
   • What is Km?
     • A constant derived from the rate constants. Under true Michaelis-Menten conditions is an estimate of the dissociation constant of E from S
     • SMALL Km MEANS TIGHT BINDING
     • BIG Km MEANS WEAK BINDING
   • What is Vmax
     • Constant at a given [E]
     • Theoretical max rate of the reaction but NEVER ACTUALLY REACHED (asymptotically approached as [S] increases)
   • What is kcat?
     • A turnover number, the number of substrate molecules converted to product/enzyme molecule/unit of time when E is saturated with S
     • k2 = kcat = Vmax/Et
   • Catalytic efficiency measures how "perfect" the enzyme is
     • Measures how the enzyme performs when [S] is low
     • kcat/Km
   • Lineweaver-Burk
     • x-intercept = -1/Km, y-intercept = 1/Vmax, slope = Km/Vmax

5. Enzyme Inhibition

   • Reversible Inhibitors
     • Can bind to and dissociate from the enzyme, often structural analogs and used as drugs
     • Noncovalent
       • Competitive
         • I binds only to E not to ES
         • I and S bind at same site
         • I does not affect catalysis
         • Michaelis Menten Effects
           • Km increases
           • Vmax is unchanged
           • Lines with different slopes but same y-intercept
       • Uncompetitive
         • I binds only to ES not to S
         • I and S bind sites are different
         • I does not affect substrate binding, but inhibits catalytic function
         • Michaelis Menten Effects
           • Km and Vmax decrease
           • Ratio of Km to Vmax is same, so slope is same
           • Parallel lines on Lineburker-Weaver plot
       • Mixed
         • I binds at E and ES
         • I and S bind at different sites
         • I binds enzyme with or without substrate
         • Binding inhibits both substrate binding and catalysis
         • Michaelis Menten Effects
           • Vmax decreases
           • Km can increase or decrease (unchanged in extreme cases of noncompetitive
           • Wacky lines, x-intercept, y-intercept and slope are all different
         • Becomes noncompetitive in extreme cases
   • Irreversible Inhibitors
     • Permanently shut off enzyme, often toxins but sometimes used as drugs
     • Covalent
   • Go over!

6. Bioenergetics

   • Most reactions are heterolytic
   • Catabolism breaks things down
   • Anabolism builds molecules up
   • Metabolic pathways are series of connected sequential enzymatic pathways
     • Compartmentalized
     • Catabolic converge on acetyl-CoA
     • Anabolic diverge from few metabolites
   • Anabolism and catabolism are not just the reverse of each other!
   • High energy molecules
     • PEP and 1,3-BPG are the highest
       • PEP
         • Largest free energy hydrolysis
         • Hydrolysis yields enol form of pyruvate which tautomerizes to the keto form
       • 1,3-BPG
         • Mixed anhydrides or acetyl phosphates
         • Bond strain, electrostatics, and resonance create high energy
     • ATP is mid level, good for transporting P groups
       • Large negative free energy change on hydrolysis due to
         • Relief of electrostatic repulsion
         • Stabilization of products by ionization
         • Stabilization of products by resonance
         • Entropy factors
         • Greater degree of hydration of prodcuts
     • Thioesters like acetyl CoA
       • Orbital overlap between carbonyl and sulfur is not as good as resonance between oxygen and carbonyl in esters
   • Free energy changes are concentration dependent
   • Redox Reactions
     • Reduced compounds serve as fuel which electrons can be stripped off of during oxidation
     • Transfer of electrons is often accompanied by the transfer of protons and hydride
     • NAD and NADP are common redox cofactors
     • Flavin cofactors use molecular oxygen as ultimate electron acceptor, tightly bound to proteins and allow single electron transfer

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