BIOCHEM Exam 2 Lecture 9

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  1. 3 catalytic strategies that are common t most enzymes
    • acid base catalysis
    • covalent catalysis
    • metal ion catalysis

    mechanisms may be used in tandem to carry out rxns
  2. Acid-Base catalysis
    • one or more residues in the active site act as proton donor or acceptor
    • involves donation of H (from acid) or acceptance of H (from base)
    • most common mechanism -used in 60% of rxns
  3. What impact would pH have on acid-base catalysis?
    • may interfere with catalysis -- cant deprotonate or protonate
    • extreme case - denaturation
    • may change the ionization states on the aa active sites
  4. covalent catalysis
    • an amino acid residue in the active site becomes temporarily attached to part of the substrate via a covalent bond
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    • the R group is usually a nucleophile
    • -Ser, Cys, His, Lys, Asp, Glu
  5. Metal ion catalysis
    involves metal ion cofactors

    • a metal ion can:
    • -facilitate the formation of a reactive nucleophile
    • -act as an electrophile to stabilize the negative charge on a reaction intermediate (hold negative in palce so rxn can occur)
    • -hold the substrate in a favorable position for catalysis
  6. Serine proteases
    • use acid base and covalent catalysis
    • cleave peptide bonds
    • includes - chymotrypsin, trypsin, elastase
    • conserved tertiary structure despite limited sequence similarity
  7. Structural components of Ser proteases
    • binding pocket - binds an amino acid side chain
    • catalytic triad - what does the chemistry
    • oxyanion hole - stabilizes the intermediate
  8. What governs substrate specificity in ser proteases? Apply to 3 types.
    the nature of aa in the binding pockets

    • Chymotrypsin
    • -deep, hydrophobic pocket

    • Trypsin
    • -deep, negative charge at bottom

    • Elastase
    • -shallow, hydrophobic pocket
  9. What makes the catalytic triad?
    • Serine: the nucleophile
    • Histidine: removes proton from Ser, makes Ser more nucleophilic
    • Aspartate: makes His imidazole side chain more electrophilic, assisting in proton removal
  10. Chymotrypsin: catalytic mechanism
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    • STEP 1
    • -protein enters the active site

    • STEP 2
    • -rxn begins w/ nucleophilic attack by the Ser oxygen on the carbonyl of the peptide
    • -nucleophilic attack produces a tetrahedral intermediate - a carbonyl w/ 4 bonds
    • -intermediate has neg. charge, stabilized by adjacent aa in oxyanion hole (H bonds with amide bond nitrogens of Gly 193 and Ser 195)

    • STEP 3
    • -the tetrahedral intermediate resolves to an acyl-enzyme intermediate 
    • --> amide side of the broken peptide bond hydrogen bonded to the His caroxyl side covalently bound to the Ser oxygen
    • -amide removes the hydrogen from the active site His and departs from the active site

    • STEP 4
    • -water diffuses into the active site and acts as a nucleophile
    • -->hydrolyzes the ester bond of the acyl-enzyme complex

    • STEP 5
    • -tetrahedral intermediate forms when Ser oxygen withdraws the hydrogen from His

    • STEP 6
    • -carboxylic acid product released
    • -Ser alcohol group restored

    • STEP 7
    • -carboxylic acid diffuses out of the active site 
    • -enzyme restored to the original config.
  11. Chymotrypsin catalytic mechanism, rxn key points
    • positioning of catalytic triad aas in active site creates the nucleophile that can cleave the peptide bond
    • ---> creates transient, covalent, acyl-enzyme intermediate
    • positioning of Gly 193 and Ser 195 form microenvironment (oxyanion hole)
    • --->stabilizes an otherwise unstable tetrahedral intermediate
    • His serves as a general acid-base catalyst
    • --->withdraws proton to increase the nucleophilicity of Ser
    • --->donates proton to resolve the 2nd tetrahedral intermediate
  12. Enolase info & mechanism
    • an enzyme that uses metal ion catalysis
    • --->catalyzes conversion of 2-phosphoglycerate to phosphoenolpyruvate
    • --->2 bound Mg2+ ions essential to mechanism
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  13. Enzymes must be regulated
    • organisms are open systems
    • organisms are not equilibrium systems
    • eukaryotes are compartmentalized creatures
    • ---> enzymes must function in teh right palce at the right time
  14. Types of enzyme regulation (5)
    • allosteric control: enzyme activity regulated through binding of small molecules at sites distinct from the active site
    • covalent modification: enzyme structure and function modified by reversible, covalent, addition of modifying group
    • isozymes: homologous (within organism) enzyme proteins that catalyze the same rxn but often with different kinetics
    • proteolytic activation: enzyme irreversibly converted from inactive precursor to active species
    • protein turnover: enzyme activity regualted by degrading enzyme proteins or by synthesizing new ones
  15. allosteric enzymes
    • act as info sensors and are frequently found in metabolic pathways that display feedback  inhibition
    • usually multi-subunit proteins regulated by allosteric modulators (effectors)
    • do not conform to Michaelis-Menten kinetics (sigmoidal curve, change in quaternary structure, coop)
    • allosteric modulators bind to regulatory allosteric sites
  16. Aspartate Transcarbamoylase (ATCase) model of allosteric control
    • catalyzes first step in pyrimidine biosynthesis
    • inhibited by cytidine triphosphate (CTP)
    • ATCase catalyzes the condensation of carbomoyl phosphate and aspartate to make N-carbamoyl aspartate and orthophosphate (Pi)
    • ATCase activity decreases as [CTP] increases - unusual because CTP is not structually similar, this suggests CTP is an allosteric inhibitor
    • ATCase has a sigmoidal kinetics curve (not MM) - at low [Asp]the reaction rate is low, suggests multiple subunits and coop
    • T and R states (T = low affinity for sub. R = high affinity for sub)
    • CTP stabilizes T state - binds to regulatory subunits (more CTP = shift to right, decrease activity, need more S for product formation)
    • ATP stabilizes R state - upregulates purines so you need more pyrimidines, opp of CTP
  17. regulatory advantages of allosteric enzymes
    • can be regulated by later intermediates or signal molecules
    • product formation can be adjusted based on the current demands of cell
  18. Covalent modification is:
    • a rapid reversible change to one or more aa residues in an enzyme
    • phosphorlyation is the most common ( catalyzed by kinases, requires use of ATP or GTP)
  19. kinases
    • removes the terminal phosphate (gamma) from ATP and add it to the hydroxyl on serine, threonine, or tyrosine
    • do not catalyze the reverse reaction (dephosphorylation uses phosphatases)
  20. regulatory advantages of phosphorylation
    • structural: addition of phosphate brings 2 (-) charges, phosphoryl group can H bond
    • timing: phosphorylation/dephsporylation cycle adjustable
    • energetics: large delta g for phosphorylation, 50kj/mol provided by hydrolysis of ATP gamma phosphate, about 1/2 of that energy is conserved in the phosphorylated protein, phosphorylation/dephosphorylation can change the equilibrium between opposing functional states
    • sensing function: using ATP as the phosphoryl donor links enzyme regulation to the energy state of the cell
    • amplification of information signals: a single protein kinase can rapidly phosph. hundreds of target proteins, which may then catalyze a huge number of rxns
    • different stages of development
    • different responses to allosteric modulators
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BIOCHEM Exam 2 Lecture 9
2014-02-16 19:08:23

Exam 2 material
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