Part 4

• Catalysis

  The general principles of catalysis and the kinetic parameters used to describe enzyme action
• Understanding the complete mechanism of action of a purified enzyme

  1. Identify all substrates, cofactors, products, and regulators

  2. Know the temporal sequence in which enzyme bound reaction intermediates form

  3. Know the structure of each intermediate and each transition state

  4. Know the rates of interconversion between intermediates

  5. Know the structural relationship of the enzyme to each intermediate

  6. Know the energy that all reacting and interacting groups contribute to the intermediate complexes and transition states
• There are only a few enzymes in which we have an understanding that meets all these requirements
• Chymotrypsin


  A protease that catalyzes the hydrolytic cleavage of peptide bonds, specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr)
• Chymotrypsin mechanism

  • It does not catalyze a direct attack of water on the peptide bond; instead, a transient covalent acyl enzyme intermediate is formed
  • The reaction has two distinct phases: acylation and deacylation
• Chymotrypsin hydrolysis of p-nitrophenylacetate

  1. Rapid burst before leveling off to a slower rate

  2. Burst phase corresponds to the release of just under one molecules of p-nitrophenol for every enzyme molecule present

  3. Burst implies that the rate limiting step of catalysis occurs after release of the product being monitored

  4. Burst reflects a rapid acylation of all the enzyme molecules, with turnover limited by a subsequent, slower deacylation step
• Chymotrypsin catalyzed cleavage


  Exhibits a bell shaped pH rate profile
• k_cat


  Reflects the ionization state of His, with the decline at low pH resulting from the protonation of His
• 1/K_m

  Reflects the ionization of the alpha amino group of Ile, which forms a salt bridge to Asp to stabilize the active conformation of the enzyme
• Chymotrypsin reaction mechanism

  1. Step 1: Substrate binding positions the peptide bond for attack

  2. Step 2: Ser and His generate a strongly nucleophilic alkoxide ion on Ser, which attacks the peptide carbonyl group, forming a tetrahedral acyl enzyme intermediate

  3. Step 3: Collapse of the tetrahedral intermediate breaks the peptide bond, with the amino leaving group protonated by His

  4. Step 4: A water molecule is deprotonated by general base catalysis, generating a strongly nucleophilic hydroxide ion

  5. Step 5: Attack of hydroxide on the ester linkage of the acyl enzyme generates a second tetrahedral intermediate

  6. Step 6: Collapse of the second tetrahedral intermediate forms the second product and regenerates the free enzyme
• New pharmaceutical agents are almost always designed to inhibit an enzyme
• Retroviruses

  Possess an RNA genome and an enzyme, reverse transcriptase, that can use RNA to direct the synthesis of a complementary DNA
• Retrovirus life cycle

  1. RNA genome is converted to duplex DNA by reverse transcriptase

  2. Duplex DNA is inserted into a chromosome in the host cell nucleus by the enzyme integrase

  3. Integrated viral genome can remain dormant or be transcribed back into RNA for translation into viral proteins

  4. Most viral genes are translated into large polyproteins, which are cut by the HIV protease into individual proteins needed to make the virus
• Major subclasses of proteases

  • Serine proteases
  • Cysteine proteases
  • Aspartyl proteases
  • Metalloproteases
• HIV protease

  An aspartyl protease with two active site Asp residues that facilitate the direct attack of a water molecule on the carbonyl group of target peptide bonds
• Viral genome integration

  1. Inserted into a chromosome in the nucleus of the host cell by the enzyme integrase

  2. The integrated copy of the viral genome can remain dormant indefinitely

  3. Alternatively, it can be transcribed back into RNA, which can then be translated into proteins to construct new virus particles
• Viral enzymes

  • Reverse transcriptase
  • Integrase
  • Protease
• Viral enzymes

  • They represent the most promising drug targets
• Protease subclasses

  • Serine proteases (e.g. chymotrypsin, trypsin)
  • Cysteine proteases
  • Aspartyl proteases
  • Metalloproteases
• HIV protease

  An aspartyl protease with two active site Asp residues that facilitate direct attack of a water molecule on the peptide bond to be cleaved
• HIV protease inhibitors

  • They form noncovalent complexes with the enzyme but bind to it so tightly that they can be considered irreversible inhibitors
  • They are designed as transition state analogs
• The HIV protease is most efficient at cleaving peptide bonds between Phe and Pro residues
• Core structure of HIV protease inhibitors

  A main chain with a hydroxyl group positioned next to a branch containing a benzyl group, which mimics the negatively charged oxygen in the tetrahedral intermediate in the normal reaction
• The availability of effective HIV protease inhibitor drugs has vastly increased the life span and quality of life of millions of people with HIV and AIDS
• In 2018, 23.3 million of the 38 million people living with HIV infection were receiving antiretroviral therapy
• Hexokinase

  A bisubstrate enzyme that catalyzes the reversible reaction of beta-d-glucose to glucose 6-phosphate
• ATP and ADP always bind to hexokinase as a complex with metal ion Mg2+
• Hexokinase reaction

  The gamma-phosphoryl of ATP is transferred to the hydroxyl of C-6 of glucose
• Hexokinase

  • It can discriminate between glucose and water because of a conformational change in the enzyme when the correct substrate binds
  • It is a good example of induced fit
• When glucose is not present, hexokinase is in an inactive conformation with the active site amino acid side chains out of position for the reaction
• When glucose, but not water, and Mg ATP bind, the binding energy induces a conformational change in hexokinase to the catalytically active form
• The binding of xylose to hexokinase is sufficient to induce a change to its active conformation, and the enzyme is thereby tricked into phosphorylating water
• Enzyme specificity is not always as simple as binding one compound but not another, it is observed in the relative rates of subsequent catalytic steps
• Hexokinase catalytic mechanism

  • It uses several mechanisms including general acid base catalysis and transition state stabilization
• Enolase

  A glycolytic enzyme that catalyzes the reversible dehydration of 2-phosphoglycerate to phosophoenolpyruvate
• Enolase reaction mechanism

  1. Lys acts as a general base catalyst, abstracting a proton from the C-2 of 2-phosphoglycerare

  2. Glu acts as a general acid catalyst, donating a proton to the -OH leaving group
• Enolase active site

  • The carboxyl group of 2-phosphoglycerate undergoes strong ionic interactions with two bound Mg2+ ions, greatly enhancing the electron withdrawal by the carbonyl and rendering the C-2 protons sufficiently acidic
  • The metal ions also act to shield the two negative charges on the carboxyl oxygen atoms that transiently exist in close proximity to each other
• Penicillin interferes with the synthesis of peptidoglycan, the major component of the rigid cells wall that protects bacteria from osmotic lysis
• Penicillin mechanism of action

  1. Penicillin binds to the active site of the transpeptidase enzyme through a segment that mimics the conformation of the D-Ala-D-Ala segment of the peptidoglycan precursor

  2. An active site Ser attacks the carbonyl of the beta-lactam ring, generating a covalent adduct between penicillin and the enzyme

  3. This irreversibly inactivates the enzyme, blocking cell wall synthesis and causing the bacterial cell to burst
• Bacteria have evolved resistance to penicillin by expressing beta-lactamases that cleave the beta-lactam ring