ORGANIC CHEMISTRY 354

ENZYMES

COMPARE TO LAB REACTIONS

CHYMOTRYPSIN

CHYMOTRYPSIN (INTERACTIVE)GIF

ENZYMES - NATURE'S CATALYSTS

EXAMPLE: CAN CATALYZE HYDROLYSIS OF AMIDES

• RCONHR' + H₂O ---> RCO₂H + R'NH₂

• IN LAB
  • BOIL IN ACID
  • BOIL IN BASE

• IN THE SMALL INTESTINE
  • PROTEINS (AMIDES) ---> RCO₂H + R'NH₂
  • GOES AT 37^o
  • GOES AT ALMOST NEUTRAL pH

  • HOW???

CATALYZED BY AN ENZYME, A PROTEIN

CHYMOTRYPSIN

• ONE OF FIRST ENZYMES AVAILABLE COMMERCIALLY
• STUDIED EXTENSIVELY
• 245 AMINO ACIDS
• THREE (3) CHAINS
• HYDROLYZES:
  • PROTEINS
  • SIMPLE AMIDES
  • SIMPLE ESTERS

• HOW? NEED SOME DATA!!

1. ADD p-NITROPHENYL ACETATE TO CHYMOTRYPSIN
   • QUENCH (STOP) BEFORE HYDROLYSIS IS COMPLETE
   • ANALYZE ENZYME
   • FIND SERINE-195 AS AN ESTER
     CHYMOTRYPSIN (INTERACTIVE)GIF

   • BUT HOW CAN THIS TRANSESTERIFICATION OCCUR AT pH = 7.4?

2. REACTS FASTEST AT pH = 7.4
   • IN LAB WE NEED ACID OR BASE
   • HYDROLYSIS OF ESTERS IS ALSO CATALYZED BY OTHER SUBSTANCES
     • IMIDAZOLE IS IN HISTIDINE
     • HISTIDINE-57 IS NEAR SERINE-195

     CHYMOTRYPSIN (INTERACTIVE)GIF

   • PROPOSED MECHANISM

   • CARBOXYLATE ANION OF ASPARTIC ACID-102 ACTIVATES IMIDAZOLE NITROGEN OF HISTIDINE-57.
   CHYMOTRYPSIN (INTERACTIVE)

3. DEVELOPING NEGATIVE CHARGE IS STABILIZED BY SERINE-195 AND GLYCINE-193.
   CHYMOTRYPSIN (INTERACTIVE)

4. ISOLEUCINE-16
   • ACYLATE ALL FREE NH₂ GROUPS
   • ENZYME IS STILL ACTIVE, UNLESS ISOLEUCINE-16 IS ACYLATED
   • AT pH 7.4, NH₂ IS NH₃⁺
     • IF CHANGE pH
       • OPTICAL ACTIVITY CHANGES
       • CONFORMATION CHANGES
     CHYMOTRYPSIN (INTERACTIVE)GIF

   • ISOLEUCINE-16 KEEPS HISTIDINE NEAR SERINE-195
     • HOW?
       • ASPARTIC ACID-194 IS ATTACHED TO SERINE-195
   CHYMOTRYPSIN (INTERACTIVE)

5. CATALYZES PEPTIDE BONDS OR ESTER BONDS BEST IN WHICH THERE IS A PHENYL GROUP ATTACHED TO THE ACYL PORTION
   • THERE IS A HYDROPHOBIC POCKET (LINED WITH NONPOLAR GROUPS)
     CHYMOTRYPSIN (INTERACTIVE)GIF

   • POCKET HOLDS SUBSTRATE IN PLACE

6. DIISOPROPYL FLUOROPHOSPHATE
   • ADD TO ENZYME
   • ENZYME IS INHIBITED
   • SERINE-195 IS BONDED TO PHOSPHOROUS
   • POISON ONLY ONE GROUP, STOP ENZYME
   SUMMARY CHYMOTRYPSIN (INTERACTIVE) GIF

   • SERINE - HYDROLYZES
   • HISTIDINE - CATALYZES
   • OTHERS - STABILIZE OR ACTIVATE
   • ISOLEUCINE - KEEPS CONFORMATION
   • HYDROPHOBIC POCKET - HOLDS SUBSTRATE
   • pH
     • WEAKER NITROGEN BASE IS A FREE BASE
     • STRONGER NITROGEN BASE IS A POSITIVE ION (NH₃⁺¹)