Question

A) Draw the dipeptide R-T at pH 7 and CLEARLY label the peptide bond to be broken in the presence of trypsin. 3pts B) Draw the five-step mechanism for the trypsin cleavage of the dipeptide. 5pts C) Label the transition state each time it is formed. 1pt D) What mechanism is used to describe this bisubstrate reaction? 1 pt

Answer 1

T = Threonine. R - Arginine Is oelectric - 3 15. point cationic 0 5.6 pH. 7 Anionic. HIN-CH-Con OH HN CH H-CO i HNO - CH - Coo- I OH HOCH-CH₂. CH₂ - CH₂ - CH2 -NH-C-NH₂ (TRI RINH Nucleophilic addition elimination I Peptide bond formation. react. OO HAN- CH-coH HN CH-coor 1-H₂O Ľ Peptide bond. - CH - coor C-terminal Z N . N terminal H3N - CH-C At pH=7 -I - R Threonine residue. Arginine residue

Enzyme Backbone Enzyme backbone Ser 3 Asp Enzyme Backbone Enzyme backbone SI C - NH Pocket o n A. Enzymes , non Region of Enzyme that Determines what substrate the Enzyme binds. - Enzyme backbone structural Changes ixona arrodhood Enzyme backbi SH I e Induced by Ime Binding & Asp 2 Sur H Enzyme backbon & Changes Electronic & Environ- amente NEI ! ОН Catalytic Triad -NH I Pocket PMP PMA structural Binding of substrate Stimulates slight charges

Enzyme- Backbone. Enzyme backbone 5 His i Ser Enzyme Backbone. Enzyme Backbone N. - tug . N-abstracts C-NM Proton RISI from k Pocket Serine's side of enzyme. Chain, creating Tutor Soops Alkoxide ion. orida 1o en Enzyme backbone Alkoxide Ion makes Nucleophilic Enzyme Attack on backbone. carbonyl & Cof Peptide & bond & I His Ser, Asp - Enzyme backbone. I Enzyme backbone -NH Bu p rit gloro dua te grissid

Enzyme backbone - His Enzyme Backbone ENZYME DAUR MONEY way o CNH Oxy anion hole. IRL pochet " Enarme backbone Peptide bond broken and N Binds to Hon Tetrahedral Intermediate to Stabilized by stit Onyanion Hol. Histidine: modens linda Enzyme backbone. Enzyne backbone- other Half of Polypeptide ser cevaleutly. p N-H h Enzyme backbone linked to Serine Enzyme backbone - Н Ν. Η Released. Ro Half of poppeptide alleased from Enzyme. R de Lug 10 Fast Phase of Catalysis Completed on 2 erdidate

- Enzyme backbone Enzyme -backbone Enzyme backbone Enzyme backbone ② Nitrogen Attacks & Proton of water. ㅜ 아 써 " H a n water enters active other site dhe (3) Hydroxide attacks on carbonyl carbon. • Enzyme Backbone S HIS -EB Oxygen Abstracts H on N- Group, no nepaste Breaking bond Oryam on liate with hole at stabilizes Tetra hedral serine. Intermediate

EB Ser NH, Hop-B E B HN / - 0 ) oc- Woda slow phase of Catalysis complete. Whole Enzyme to original state Enzyme Backbone His Enzyme backbone. Ename backbone ser OH Enzyme backbone Cycle complete . Enzyme to start Anew e second fragment released Released

Serine Protease Catalysis • Binding of substrate si pochet a Formation of alkoxide ion o Nucleo philic attack Stabilization of intermediate o Breakage of peptide bond. - • Release of peptide 1 • Entry and activation of water • Release of peptide 2 from encyme.
Trypsin is a good example of the use of covalent modification as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis.

This chemical modification reaction suggested that this unusually reactive serine residue plays a central role in the catalytic mechanism of trypsin