multi-substrate reactions

• Multi-substrate reactions

  Reactions with multiple substrates and products
• Michaelis-Menten kinetics

  Can describe simple single substrate reactions
• What is the feature of Km in multi substrate reactions

  Each substrate will have its own Km
• How is a Pseudo-Michaelis-Menten graph produced

  
  

  Constant concentration of S1 in excess and change S2. Lineweaver-Buk plot to determine Km for S2.

  Repeat by changing S1 but keeping S2 constant and in excess.
• Possible reaction mechanisms for multi-substrate reactions

  • Both substrates bind at once to form a ternary complex, catalysis occurs after both substrates bind
  • Substrates bind and leave sequentially, catalysis occurs after first substrate binds
• Sequential displacement reactions

  Reactions where a ternary complex forms. They can be Ordered or Disordered.
• Double displacement (ping-pong) reactions

  Characterised by formation of a substituted enzyme intermediate (E')
• Lineweaver-Burk plot for sequential displacement reactions

  • Similar to mixed non-competitive inhibition, but increasing S2 decreases slope and Vmax increases & KM decreases
• Lineweaver-Burk plot for double displacement reactions

  • Similar to uncompetitive inhibition, increasing S2 decreases intercepts and apparent Vmax & KM increase
• Chymotrypsin

  A proteolytic enzyme used for protein digestion in animals, cleaves peptide bonds selectively on C-terminal side of large hydrophobic aromatic amino acids
• Chymotrypsin mechanism

  Double displacement mechanism
• Describe the sturcture of the Chymotrypsin catalytic triad

  Aspartate 102 is hydrogen bonded to histidine 57 which is also hydrogen bonded to serine 195.
• Role of catalytic triad residues

  Histidine 57 and Aspartate 102 increase the reactivity of Serine 195.

  Histidine 57 polarises the Serine 195 Hydroxyl group [Alkoxide ion] by acting as a general base catalyst.

  Serine 195 is becomes an alkoxide ion allowing it to act as a nucleophile.

  Aspartate 102 positions histidine 57 and counters the positive charge.
• What reaction mechanism is this

  Ordered sequential displacement.

  
• What mechanism is this

  Double displacement.

  
• Which amino acids does chymotrypsin cleave at

  Tryptophan, Tyrosine, Phenylalanine and methionine
• What is the chymotrypsin cleavage equation

  Protein + H2O = Peptide 1 + Peptide 2
• Peptide hydrolysis by chymotrypsin

  1. Protein Residue 1 has hydrophobic interactions that bind to the serine 195

  2. Histidine 57 acts as a base to Serine 195 [proton abstraction]

  3. Forms alkoxide ion on Serine 195

  4. Serine 195 acts as a nucleophile and attacks the peptide carbonyl group

  5. Unstable tetrahedral transition state formed

  6. Histidine 57 acts as an acid to the C-terminal NH group of the peptide

  7. Peptide bond is cleaved to form acyl-enzyme intermediate

  8. Histidine 57 binds water [Proton abstraction]

  9. Hydroxyl ion attacks carbonyl group of intermediate

  10. Unstable tetrahedral transition state

  11. Histidine 57 donates a proton to serine 195

  12. Peptide bond is cleaved
• How are catalytic residues identified

  Using irreversible inhibitors
• What are the classifications of irreversible inhibitors

  Group-specific

  Suicide

  Affinity labels
• What are group-specific reagents

  They react with specific residues
• What are affinity labels

  Substrate analogues that bind to the active site irreversibly
• What are suicide inhibitors

  Substrate analogues that are converted by the enzyme into affinity labels.
• What type of inihbitor is 5-fluorouracil

  Suicide and irreversible
• How was the Serine 195 residue identified in chymotrypsin

  Using DIPF which is an OH group specific reagent.

  DIPF only modified Serine 195 meaning that this is the most reactive serine residue in chymotrypsin.
• What type of protease is chymotrypsin

  Serine due to the critical serine 195 residue in the active site
• How was the Histidine 57 residue in chymotrypsin identified

  Using TPCK which is an affinity label. TPCK has additional chloromethyl ketone groups than the substrate. This is known as the reactive group and forms a covalent bond with the histidine 57 residue therefore, this is the most reactive histidine residue in chymotrypsin.
• what is convergent evolution

  When the same structure evolves independently more than once.
• what are the three evoluntinonary types of the catalytic triad

  Serine proteases.

  Subtilisin-like proteases.

  Wheat carboxypeptidase II.
• What resides does wheat carboxypeptidase use

  Histidine and Cysteine
• What are the main serine proteases

  Trypsin, Elastase and chymotrypsin.
• What si site-directed mutagenesis

  The insertion, deletion or addition of a base (s) in order to alter the enzymes properties
• Where does trypsin cleave

  After long positively charged residues such as lysine and arginine
• Where does elastase cleave

  After small residues such as alanine and serine.
• What amino acid is in the trypsin active site

  Aspartate 189 instead of serine 195 like chymotrypsin. This provides a negative charge to attract positively charge residues on arginine and lysine
• What residues does elastase have in the active site

  Valine 216 and valine 190 block access from large amino acids.
• What determines serine-protease substrate specificity

  The pocket residues
• What are pocket residues

  These are the residues that line the substrate binding pocket