Chapt. 8 Enzymes as catalysts

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Chapt. 8 Enzymes as catalysts

• Ch. 8 Enzymes as catalysts
• Student Learning Outcomes
• Explain general features of enzymes as catalysts
  Substrate -gt Product
• Describe nature of catalytic sites
• general mechanisms
• Describe how enzymes lower activation energy of
  reaction
• Explain how drugs and toxins inhibit enzymes
• Describe 6 categories of enzymes

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Catalytic power of enzymes

• Enzymes do not invent new reactions
• Enzymes do not change possibility of reaction to
  occur (energetics)
• Enzymes increase the rate of reaction by factor
  of 1011 or higher

Fig. 8.1 box of golfballs, effect of browning
enzyme
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Enzymes catalyze reactions

• Enzymes provide speed, specificity and
  regulatory control to reactions
• Enzymes are highly specific for biochemical
  reaction catalyzed (and often particular
  substrate)
• Enzymes are usually proteins
• (also some RNAs ribozymes)
• E S ? ES binding substrate
• ES ? EP substrate converted to bound product
• EP ? E P release of product

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Glucokinase is a typical enzyme

• Glucokinase is typical enzyme
• ATP D-glucose 6-phosphotransferase
• Very specific for glucose
• Not phosphorylate other hexoses
• Only uses ATP, not other NTP
• 3D shape of enzyme critical for its function
  (derived from aa sequence)

Fig. 8.2 glucokinase
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A. Active site of enzyme

• Enzyme active site does catalysis
• Substrate binds cleft formed by aa of enzyme
• Functional groups of enzyme, also cofactors bond
  to substrate, perform the catalysis

Fig. 8.4
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B. Binding site specificity

• Substrate binding site is highly specific
• Lock-and-key model 3D shape recognizes
  substrate (hydrophobic, electrostatic, hydrogen
  bonds)
• Induced-fit model enzyme conformational change
  after binding substrate
• galactose differs from
• glucose, needs separate
• galactokinase

Fig. 8.5 glucokinase
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Glucokinase conformational change

• Conformation change of glucokinase on binding
  glucose
• Binding positions substrate to promote reactions
• Large conformational change adjusts actin fold,
  and facilitates ATP binding
• Actin fold named for G-actin
• (where first described Fig. 7.8)

Fig. 8.6 glucokinase (Yeast hexokinase)
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Transition state complex

• Energy Diagram substrates are activated to
  react
• Activation energy barrier to spontaneous
  reaction
• Enzyme lowers activation energy
• Transition-state complex is stabilized by diverse
  interactions

Fig. 8.7
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Transition-state complex

• Transition-state complex binds enzyme tightly
• transition-state analogs are potent inhibitors
  of enzymes (more than substrate analogs)
• make prodrugs that convert to active analogs at
  site of action
• Abzymes catalytic antibodies that have aa in
  variable region like active site of transition
  enzyme
• Artificial enzymes catalyze reaction
• Ex. Abzyme to Cocaine esterase destroys cocaine
  in body

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II. Catalytic mechanism of chymotrypsin -
example enzyme

• Chymotrypsin, serine protease, digestive enzyme
• Hydrolyzes peptide bond (no reaction without
  enzyme)
• Serine forms covalent intermediate
• Unstable oxyanion (O-) intermediate
• Cleaved bond is
• scissile bond

Fig. 8.8
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B. Catalytic mechanism of chymotrypsin

• 1. Specificity of binding
• Tyr, Phe, Trp on denatured proteins
• Oxyanion tetrahedral intermediate
• His57, Ser195, Asp
• 2. acyl-enzyme intermediate
• 3. Hydrolysis of acyl-enzyme intermediate

Fig. 8.9
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Mechanism of chymotrypsin, cont.

• 3. Hydrolysis of acyl-enzyme intermediate
• Released peptide product
• Restores enzyme

Fig. 8.9
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Energy diagram revisited with detail

• Chymotrypsin reaction has several transitions
• See several steps
• Lower energy barrier to uncatalyzed

Fig. 8.10
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III. Functional groups in catalysis

• Functional groups in catalysis
• All enzymes stabilize transition state by
  electrostatic
• Not all enzymes form covalent intermediates
• Some enzymes use aa of active site (Table 1)
• Ser, Lys, His - covalent links
• His - acid-base catalysis
• peptide backbone NH stabilize anion
• Others use cofactors (nonprotein)
• Coenzymes (assist, not active on own)
• Metal ions (Mg2, Zn2, Fe2)
• Metallocoenzymes (Fe2-heme)

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Coenzymes assist catalysis

• Activation-transfer coenzymes
• Covalent bond to part of substrate enzyme
  completes
• Other part of coenzyme binds to the enzyme
• Ex. Thiamine pyrophosphate is derived from
  vitamin thiamine
• works with many different enzymes
• enzB takes H from TPP carbanion attacks keto
  substrate, splits CO2

Fig. 8.11
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Other activation-transfer coenzymes

• Activation-transfer coenzymes
• Specific chemical group binds enzyme
• Other functional group participates directly in
  reaction
• Depends on enzyme for specificity of substrate,
  catalysis

Fig. 8.12 A CoA forms thioesters with many acyl
groups acetyl, succinyl, fatty acids
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Oxidation-reduction coenzymes

• Oxidoreductase enzymes use other coenzymes
• Oxidation is loss of electrons (loss H, or gain
  O)
• Reduction is gain electrons (gain H, loss of O)
• Redox coenzymes do not form covalent bond to
  substrate
• Unique functional groups
• NAD (and FAD) special
• role for ATP generation
• Ex. Lactate dehydrogenase
• oxidizes lactate to pyruvate
• transfers e- H to NAD
• -gt NADH

Fig. 8.13 lactate dehydrogenase
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Metal ions assist in catalysis

• Positive metal ions attract electrons contribute
• Mg2 often bind PO4, ATP ex. DNA polymerases
• Some metals bind anionic substrates

• Fig. 8.14
• ADH alcohol dehydrogenase
• oxidizes alcohol to acetaldehyde
• and NAD to NADH
• Zn2 assists with NAD
• (In Lactate dehydrogenase, a His residue assisted
  the reaction)

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pH affects enzyme activity

• Each enzyme has characteristic pH optimum
• Depends on active-site amino acids
• Depends on H bonds required for 3D structure
• Each enzyme has optimum
• temperature for activity
• Humans 37oC
• Taq polymerase
• for PCR 72oC

Fig. 8.15 optimal pH for enzyme
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V. Mechanism-based inhibitors

• Inhibitors decrease rate of enzyme reaction
• Mechanism-based inhibitors mimic or participate
  in intermediate step of reaction
• Covalent inhibitors
• Transition-state analogs
• Heavy metals

Fig. 8.2 organophosphate inhibitors include two
insecticides, and nerve gas Sarin
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Covalent inhibitors

• Covalent inhibitors form covalent or very tight
  bonds with functional groups in active site

Fig. 8.16 DFP di-isopropylfluorophosphate
prevents acetylcholinesterase from degrading
acetylcholine
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Transition state analogs

• Transition-state analogs bind more tightly to
  enzyme than substrate or product
• Penicillin inhibits glycopeptidyl transferase,
  enzyme that synthesizes cross-links in bacterial
  cell wall.
• Kills growing cells by inactivating enzyme

Fig. 8.17 penicillin
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Allopurinol treats gout

• Allopurinol is suicide inhibitor of xanthine
  oxidase
• Treatment for gout (decreases formation of
  urate)

Fig. 8.18
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Basic reactions and classes of enzymes

• 6 basic classes of enzymes
• Oxidoreductases
• Oxidation-reduction reactions (one gains, one
  loses e-)
• Transferases
• Group transfer functional group from one to
  another
• Hydrolases cleave C-O, C-N and C-S bonds
• addition of H2O in form of OH- and H
• Lyases diverse cleave C-C, C-O, C-N
• Isomerases rearrange, create isomers of starting
  
• Ligases synthesize C-C, C-S, C-O and C-N bonds
• Reactions often use cleavage of ATP or others

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Some example enzymes

• Example enzymes
• Group transfer
• transamination
• transfer of amino group
• Isomerase
• rearranges atoms
• ex. In glycolysis

Fig. 8.19
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Key concepts

• Enzymes are proteins (or RNA) that are catalysts
• accelerate rate of reaction
• Enzymes are very specific or substrate
• Enzymes lower energy of activation to reach
  high-energy intermediate state
• Functional groups at active site (amino acid
  residues, metals, coenzymes) cause catalysis
• Mechanisms of catalysis include acid-base,
  formation covalent intermediates, transition
  state stabilization

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Review questions

• 4. The reaction shown fits into which
  classification?
• Group transfer
• Isomerization
• Carbon-carbon bond breaking
• Carbon-carbon bond formation
• Oxidation-reduction
• 5. The type of enzyme that catalyzes
• this reaction is which of the following?
• Kinase
• Dehydrogenase
• Glycosyltransferase
• Transaminase
• isomerase