PROTEASE-CATALYZED PEPTIDE SYNTHESIS

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1 RTEAE-CATALYZED ETIDE YTEI Thermodynamic approach RC R' RC R' RCR' + 2 endergonic; requires manipulation of conditions (organic cosolvents, solubility of products, temperature, etc.) Kinetic approach 2 RC 2 RC 2 R' + E RC 2 R' E R C XE R' R" 2 RCR" faster than thermodynamic approach, but limited to enzymes that form acyl-enzyme intermediates Transpeptidation RCR' + 2 R" RCR" + 2 R' Cell-free protein synthesis in vitro transcription/translation using E. coli (or rabbit reticulocyte) extracts containing all proteins and RAs needed for protein synthesis

2 EZYMATIC YTEI F AARTAME Aspartame is a low-calorie sweetener ( times sweeter than sucrose) whose synthesis has attracted much attention. An enzymatic approach has certain advantages over a conventional chemical process in which the required stereospecificity adds to the production costs. Thermolysin is a zinc protease and its use in aspartame synthesis is an example of the thermodynamic approach. Z C + 2 h C 3 thermolysin Z C h C 3 hydrogenation L-Asp-L-he-C 3!-Aspartame L-Z-Asp D,L-he-C3 L-Z-Asp-L-he-C 3 + where Z = benzyloxycarbonyl 2 h C 3 racemization D,L-he-C 3 D-he-C 3 EZYMATIC CVERI F RCIE IT UMA IULI emisynthetic human insulin has been produced enzymatically from porcine insulin on an industrial scale to satisfy the demand of millions of diabetics worldwide. Trypsin is a serine protease that cleaves specifically after basic amino acids. This is an example of the kinetic approach. Gly 1 he 1 Lys 29 -Ala 30 IULI (porcine) -Ala- TRYI Gly 1 he 1 Lys 29 des-ala B30 -IULI -Thr-Bu t TRYI Gly 1 he 1 Lys 29 -Thr 30 -Bu t IULI-t-butylester CF 3 C Gly 1 he 1 Lys 29 -Thr 30 IULI (human)

3 YILGICALLY IMRTAT RTEAE erine roteases Trypsin Chymotrypsin Elastase Thrombin Factor Xa Cleavage site Arg-Xaa, Lys-Xaa Tyr-Xaa, he-xaa, Trp-Xaa Val-Xaa Arg-Gly Arg-Ile, Arg-Gly Function Tissue degradation Blood clotting Blood clotting Aspartyl roteases epsin Renin Cathepsin D Chymosin IV-rotease Cleavage site he-he, he-tyr, etc. Leu-Val he-he, Leu-Leu, etc. he-met he-ro, Tyr-ro, etc. Function Blood pressure regulation Tissue degradation Milk clotting Viral replication Metallo-roteases Carboxypeptidase ACE E ECE Collagenase tromelysin Cleavage site Xaa-Tyr, Xaa-he he-is, he-leu, ro-he he-leu, Cys-he Trp-Val Gly-Leu, Gly-Ile Gly-Leu, Gly-Ile Function Converts angiotensin I to angiotensin II Multifunctional (e.g. enkephalin) Converts Big-endothelin to endothelin Extracellular matrix modification Extracellular matrix modification

4 ERIE RTEAE IIBITR Types of inhibitor irreversible reversible Functional groups Chloromethylketone ulfonylfluoride Ester Boric acid Aldehydes Ketones Trifluoromethylketones α-ketoacids -CC 2 Cl - 2 F -CR -B() 2 -C -CR (R= alkyl, aryl) -CCF 3 -CC Thrombin inhibitors AA (racemate) K i = 6 nm IC 50 = 180 nm C Elastase inhibitors Ro K i = 0.27 nm Cl ICI CF 3 2 Ar CF 3 Ar = p-fluorophenyl- K i = 100 nm

5 AARTYL RTEAE IIBITR epstatin Iva Val Val ta Ala ta ' ' ' IV-1 rotease inhibitors C 2 Me JG 365, K i = 0.66 nm CtBu C 2 Indinavir, K i = 0.52 nm aquinavir K i < 0.12 nm, IC 50 < 0.4 nm DM-323, K I = 0.27 nm

6 METALL-RTEAE IIBITR Zn 2+ Typical Zn 2+ ligands R - Zn E E I - - R - C 2 R - R R ACE inhiibtors C captopril K i = 1.7 nm IC 50 = 23 nm EtC C enalpril (prodrug) Collagenase inhibitors Ac - Et IC 50 = 70 nm Ac IC 50 = 10 nm IC 50 = 5 nm