天然水解酶擁有良好的催化能力及選擇性，但大多數的水解酶受限於環境的pH值及溫度，只能在特定的條件下進行催化反應。近年來，各種人工水解酶被設計以模擬天然水解酶的催化活性及選擇性，本研究中我們設計一系列由聚脯胺酸序列及自組裝胜肽構成的人工水解酶，其中聚脯胺酸中帶有常見於天然水解酶活性中心的催化二聯體或三聯體 而自組裝胜肽MAX1序列則可以在pH 9.0以上時形成β-sheet結構。此外，我們也合成 含脯胺酸衍生物的胜肽，試圖研究這些衍生物對胜肽結構的影響與催化效率間的關聯性。我們以CD光譜觀察不同pH下結構差異及穩定性，透過穿透式電子顯微鏡發現可以自組裝成大型柱狀或網狀結構。催化效率的部分則是以UV-Vis光譜量測，透過催化產物於特定波長下吸收度隨時間改變量判定水解催化速率，並且使用酯類、磷酯類和磷酸酯類三種不同的受質，觀察不同官能基的催化效率差異。我們發現人工水解酶的結構穩定性對催化效率有很大的影響，不同胜肽序列的設計也會因催化二聯體和三聯體的距離及立體障礙而有不同的催化速度。我們設計的人工水解酶對酯類受質有不錯的水解催化效率，而對於較難水解的磷酯類和磷酸酯類受質也有些許的催化效果。本研究說明形成穩定的二級結構為提升催化效率的關鍵，模擬天然水解酶的催化中心能夠順利進行催化反應，而可催化多種不同受質的能力使其有潛力進行更深度的應用。

Natural enzymes have great catalytic activity and selectivity, but most of them are vulnerable to environmental conditions, such as pH value and temperature. Recently, different types of peptide-based catalysts have been created and constructed to mimic the activity and specificity of natural enzymes. In this study, we designed a series of hydrolases by combining the catalytic polyproline fragment and the self-assembly peptide (MAX1), which can form β-sheet structures above pH 9.0. In addition, we also incorporated proline derivatives into peptides in an attempt to study the relationship between the peptide structure and catalytic efficiency. We measured the secondary structures of the peptides by circular dichroism spectroscopy and the electron microscopy showed that they could form nanofibers under specific conditions. We evaluated their catalytic efficiency by UV-Vis spectroscopy through monitoring the time-dependent absorbance. The substrates containing an ester bond, a phosphoester bond or a phosphate bond were used to observe the catalytic activity of different functional groups. We found that the stability of the structure played an essential role in determining the catalytic efficiency. The design of the peptide sequence might cause different catalytic ability due to the distances of catalytic dyad/triad or steric effects. The artificial hydrolase enhanced the catalytic activity on ester hydrolysis significantly and also showed slight activity on both phosphoester and phosphate hydrolysis. In conclusion, the formation of stable secondary structures influenced the catalytic efficiency significantly. The mimic of catalytic activities of natural enzymes was successful, and the ability to catalyze a variety of different substrates made it potential for deeper applications.

中文摘要 I
Abstract II
誌謝辭 III
目錄 IV
圖目錄 VII
表目錄 XII
第一章 緒論 1
1-1 聚脯胺酸(Polyproline) 1
1-2 PPII(Polyproline II) 結構 2
1-3 立體電子效應(Stereoelectronic effect) 2
1-4 β-sheet 結構 5
1-5 水解反應(Hydrolysis reaction) 6
1-6 天然水解酶作用機制 9
1-7 人工水解酶的設計 12
1-8 研究動機 16
第二章 實驗部分 17
2-1 實驗儀器 17
2-2 實驗藥品 18
2-3 脯胺酸衍生物合成 22
2-3-1 合成步驟 22
2-3-2 合成方法 25
2-4 固相胜肽合成法 (Solid phase peptide synthesis, SPPS) 32
2-4-1 酯化 (esterification)、醯胺化反應 (amidation) 34
2-4-2 去保護 (deprotection) 34
2-4-3 活化 (activation) 35
2-4-4 耦合 (coupling) 35
2-4-5 切除 (cleavage) 36
2-5 胜肽之合成、純化與鑑定 37
2-6 圓二色光譜儀 (Circular dichroism spectrometer, CD) 39
2-7 光譜測量 43
2-7-1 UV光譜測量胜肽濃度 43
2-7-2 Far-UV 光譜測量 43
2-7-3 ATR-FTIR 光譜測量 43
2-7-4 催化水解實驗 44
2-8 催化活性數據處裡 49
2-9 穿透式電子顯微鏡 (Transmission electron microscope) 51
2-10 胜肽結構之理論計算模擬 51
第三章 結果與討論 53
第一部分 自組裝胜肽作為人工水解酶之結構探討 53
3-1自組裝人工水解酶之設計 53
3-2自組裝胜肽二級結構鑑定 56
3-3自組裝之TEM鑑定 62
3-4 衰減全反射式霍式轉換紅外光譜(ATR-FTIR) 66
第二部分 自組裝胜肽對酯類水解催化測定 69
3-5自組裝胜肽對酯類水解催化效率探討 69
3-5-1 pH 7.4下自組裝胜肽對酯類水解催化效率之探討 69
3-5-2 pH 9.0下自組裝胜肽對酯類水解催化效率之探討 71
3-5-3 pH 10.0下自組裝胜肽對酯類水解催化效率之探討 73
3-5-4分子模擬自組裝胜肽結構 76
3-5-5相關研究酯類水解催化效率比較 79
第三部分 自組裝胜肽對磷酯類與磷酸酯類水解催化測定 80
3-6自組裝胜肽對磷酯類水解催化效率探討 80
3-7自組裝胜肽對磷酸酯類水解催化效率探討 85
3-7-1 自組裝胜肽催化水解p-NPP之探討 85
3-7-2 相關研究磷酸酯類水解催化效果探討 89
第四章 結論與展望 90
參考文獻 92
附錄 98

1. Ruggiero, M. T.; Sibik, J.; Orlando, R.; Zeitler, J. A.; Korter, T. M. Measuring the Elasticity of Poly-L-Proline Helices with Terahertz Spectroscopy. Angew. Chem. Int. Ed. 2016, 55 (24), 6877-81.
2. Traub, W.; Shmueli, U. Structure of Poly-L-Proline I. Nature 1963, 198, 1165-1166.
3. Kakinoki, S.; Hirano, Y.; Oka, M. On the Stability of Polyproline-I and II Structures of Proline Oligopeptides. Polym. Bull. 2004, 53 (2), 109-115.
4. Moradi, M.; Babin, V.; Roland, C.; Darden, T. A.; Sagui, C. Conformations and Free Energy Landscapes of Polyproline Peptides. Proc. Natl. Acad. Sci. USA. 2009, 106 (49), 20746-20751.
5. Bochicchio, B.; Tamburro, A. M. Polyproline II Structure in Proteins: Identification by Chiroptical Spectroscopies, Stability, and Functions. Chirality 2002, 14 (10), 782-92.
6. Shi, Z.; Chen, K.; Liu, Z.; Kallenbach, N. R. Conformation of the Backbone in Unfolded Proteins. Chem. Rev. 2006, 106 (5), 1877-1897.
7. Hinderaker, M. P.; Raines, R. T. An Electronic Effect on Protein Structure. Protein Sci. 2003, 12 (6), 1188-94.
8. Newberry, R. W.; Raines, R. T. The n→π* Interaction. Acc. Chem. Res. 2017, 50 (8), 1838-1846.
9. Lesarri, A.; Mata, S.; Cocinero, E. J.; Blanco, S.; Lopez, J. C.; Alonso, J. L. The Structure of Neutral Proline. Angew. Chem. Int. Ed. 2002, 41 (19), 4673-4676.
10. Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines, R. T. Code for Collagen's Stability Deciphered. Nature 1998, 392 (6677), 666-667.
11. Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. Conformational Stability of Collagen Relies on a Stereoelectronic Effect. J. Am. Chem. Soc. 2001, 123 (4), 777-778.
12. Taylor, C. M.; Hardré, R.; Edwards, P. J. B. The Impact of Pyrrolidine Hydroxylation on the Conformation of Proline-Containing Peptides. J. Org. Chem. 2005, 70 (4), 1306-1315.
13. Cheng, P. N.; Liu, C.; Zhao, M.; Eisenberg, D.; Nowick, J. S. Amyloid Beta-Sheet Mimics That Antagonize Protein Aggregation and Reduce Amyloid Toxicity. Nat. Chem. 2012, 4 (11), 927-33.
14. Díaz-Caballero, M.; Navarro, S.; Nuez-Martínez, M.; Peccati, F.; Rodríguez-Santiago, L.; Sodupe, M.; Teixidor, F.; Ventura, S. pH-Responsive Self-Assembly of Amyloid Fibrils for Dual Hydrolase-Oxidase Reactions. ACS Catal. 2020, 11 (2), 595-607.
15. Diaz-Caballero, M.; Navarro, S.; Ventura, S. Functionalized Prion-Inspired Amyloids for Biosensor Applications. Biomacromolecules 2021, 22 (7), 2822-2833.
16. Smith, P.; Nair, P. A.; Das, U.; Zhu, H.; Shuman, S. Structures and Activities of Archaeal Members of the LigD 3'-Phosphoesterase DNA Repair Enzyme Superfamily. Nucleic Acids Res. 2011, 39 (8), 3310-20.
17. Roberts, I.; Urey, H. C. The Mechanisms of Acid Catalyzed Ester Hydrolysis, Esterification and Oxygen Exchange of Carboxylic Acids. J. Am. Chem. Soc. 1939, 61 (10), 2584-2587.
18. Jencks, W. P.; Carriuolo, J. General Base Catalysis of Ester Hydrolysis1. J. Am. Chem. Soc. 1961, 83 (7), 1743-1750.
19. Bruice, T. C.; Schmir, G. L. Imidazole Catalysis. I. The Catalysis of the Hydrolysis of Phenyl Acetates by Imidazole. J. Am. Chem. Soc. 1957, 79 (7), 1663-1667.
20. Kirsch, J. F.; Jencks, W. P. Base Catalysis of Imidazole Catalysis of Ester Hydrolysis. J. Am. Chem. Soc. 1964, 86 (5), 833-837.
21. Polgar, L. The Catalytic Triad of Serine Peptidases. Cell. Mol. Life Sci. 2005, 62 (19-20), 2161-72.
22. Gutfreund, H.; Sturtevant, J. M. The Mechanism of the Reaction of Chymotrypsin with P-Nitrophenyl Acetate. Biochem. J. 1956, 63 (4), 656-661.
23. Dvir, H.; Silman, I.; Harel, M.; Rosenberry, T. L.; Sussman, J. L. Acetylcholinesterase: From 3D Structure to Function. Chem. Biol. Interact. 2010, 187 (1-3), 10-22.
24. Clark, J. D.; Schievella, A. R.; Nalefski, E. A.; Lin, L.-L. Cytosolic Phospholipase A2. J. Lipid Mediators Cell Signal. 1995, 12 (2), 83-117.
25. Proteases and Protease Inhibitors in Tumor Progression. In Chemistry and Biology of Serpins. Frank C. C.; Dennis D. C.; David G.; Maureane H.; Stuart R. S.; and Douglas M. T., Eds.; Springer, 1997; pp 89-98.
26. Gorrell, Mark D. Dipeptidyl Peptidase Iv and Related Enzymes in Cell Biology and Liver Disorders. Clin. Sci. 2005, 108 (4), 277-292.
27. Wilson, C.; Cooper, N. J.; Briggs, M. E.; Cooper, A. I.; Adams, D. J. Investigating the Breakdown of the Nerve Agent Simulant Methyl Paraoxon and Chemical Warfare Agents Gb and Vx Using Nitrogen Containing Bases. Org. Biomol. Chem. 2018, 16 (47), 9285-9291.
28. Casida, J. E.; Quistad, G. B. Organophosphate Toxicology:  Safety Aspects of Nonacetylcholinesterase Secondary Targets. Chem. Res. Toxicol. 2004, 17 (8), 983-998.
29. Liu, Y.; Moon, S.-Y.; Hupp, J. T.; Farha, O. K. Dual-Function Metal–Organic Framework as a Versatile Catalyst for Detoxifying Chemical Warfare Agent Simulants. ACS Nano 2015, 9 (12), 12358-12364.
30. Bajda, M.; Wieckowska, A.; Hebda, M.; Guzior, N.; Sotriffer, C. A.; Malawska, B. Structure-Based Search for New Inhibitors of Cholinesterases. Int. J. Mol. Sci. 2013, 14 (3), 5608-32.
31. Santarpia, L.; Grandone, I.; Contaldo, F.; Pasanisi, F. Butyrylcholinesterase as a Prognostic Marker: a Review of the Literature. J. Cachexia Sarcopenia Muscle 2013, 4 (1), 31-9.
32. Khersonsky, O.; Tawfik, D. S. The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases. J. Biol. Chem. 2006, 281 (11), 7649-56.
33. Mody, V.; Ho, J.; Wills, S.; Mawri, A.; Lawson, L.; Ebert, M.; Fortin, G. M.; Rayalam, S.; Taval, S. Identification of 3-Chymotrypsin Like Protease (3CLPro) Inhibitors as Potential Anti-SARS-CoV-2 Agents. Commun. Biol. 2021, 4 (1), 93.
34. Ferreira, J. C.; Fadl, S.; Villanueva, A. J.; Rabeh, W. M. Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease. Front. Chem. 2021, 9, 692168.
35. Dodson, G.; Wlodawer, A. Catalytic Triads and Their Relatives. Trends Biochem. Sci. 1998, 23 (9), 347-352.
36. Corey, D. R.; Craik, C. S. An Investigation into the Minimum Requirements for Peptide Hydrolysis by Mutation of the Catalytic Triad of Trypsin. J. Am. Chem. Soc. 1992, 114 (5), 1784-1790.
37. Simon, L.; Goodman, J. M. Enzyme Catalysis by Hydrogen Bonds: The Balance between Transition State Binding and Substrate Binding in Oxyanion Holes. J. Org. Chem. 2010, 75 (6), 1831-40.
38. Naray-Szabo, G.; Olah, J.; Kramos, B. Quantum Mechanical Modeling: a Tool for the Understanding of Enzyme Reactions. Biomolecules 2013, 3 (3), 662-702.
39. Goettig, P.; Brandstetter, H.; Magdolen, V. Surface Loops of Trypsin-Like Serine Proteases as Determinants of Function. Biochimie 2019, 166, 52-76.
40. Duncan, K. L.; Ulijn, R. V. Short Peptides in Minimalistic Biocatalyst Design. Biocatal. 2015, 1 (1), 67-81.
41. Bezer, S.; Matsumoto, M.; Lodewyk, M. W.; Lee, S. J.; Tantillo, D. J.; Gagne, M. R.; Waters, M. L. Identification and Optimization of Short Helical Peptides with Novel Reactive Functionality as Catalysts for Acyl Transfer by Reactive Tagging. Org. Biomol. Chem. 2014, 12 (9), 1488-94.
42. Matsumoto, M.; Lee, S. J.; Gagne, M. R.; Waters, M. L. Cross-Strand Histidine-Aromatic Interactions Enhance Acyl-Transfer Rates in Beta-Hairpin Peptide Catalysts. Org. Biomol. Chem. 2014, 12 (43), 8711-8.
43. Zastrow, M. L.; Peacock, A. F.; Stuckey, J. A.; Pecoraro, V. L. Hydrolytic Catalysis and Structural Stabilization in a Designed Metalloprotein. Nat. Chem. 2011, 4 (2), 118-23.
44. Wang, P. S.; Nguyen, J. B.; Schepartz, A. Design and High-Resolution Structure of a Beta3-Peptide Bundle Catalyst. J. Am. Chem. Soc. 2014, 136 (19), 6810-3.
45. Zhang, Q.; He, X.; Han, A.; Tu, Q.; Fang, G.; Liu, J.; Wang, S.; Li, H. Artificial Hydrolase Based on Carbon Nanotubes Conjugated with Peptides. Nanoscale 2016, 8 (38), 16851-16856.
46. Zaramella, D.; Scrimin, P.; Prins, L. J. Self-Assembly of a Catalytic Multivalent Peptide-Nanoparticle Complex. J. Am. Chem. Soc. 2012, 134 (20), 8396-9.
47. Solomon, L. A.; Kronenberg, J. B.; Fry, H. C. Control of Heme Coordination and Catalytic Activity by Conformational Changes in Peptide-Amphiphile Assemblies. J. Am. Chem. Soc. 2017, 139 (25), 8497-8507.
48. Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stohr, J.; Smith, T. A.; Hu, X.; DeGrado, W. F.; Korendovych, I. V. Short Peptides Self-Assemble to Produce Catalytic Amyloids. Nat. Chem. 2014, 6 (4), 303-9.
49. Song, R.; Wu, X.; Xue, B.; Yang, Y.; Huang, W.; Zeng, G.; Wang, J.; Li, W.; Cao, Y.; Wang, W.; Lu, J.; Dong, H. Principles Governing Catalytic Activity of Self-Assembled Short Peptides. J. Am. Chem. Soc. 2019, 141 (1), 223-231.
50. Wang, M.; Lv, Y.; Liu, X.; Qi, W.; Su, R.; He, Z. Enhancing the Activity of Peptide-Based Artificial Hydrolase with Catalytic Ser/His/Asp Triad and Molecular Imprinting. ACS Appl. Mater. Interfaces 2016, 8 (22), 14133-41.
51. Hung, P. Y.; Chen, Y. H.; Huang, K. Y.; Yu, C. C.; Horng, J. C. Design of Polyproline-Based Catalysts for Ester Hydrolysis. ACS Omega 2017, 2 (9), 5574-5581.
52. Ting, Y. H.; Chen, H. J.; Cheng, W. J.; Horng, J. C. Zinc(Ii)-Histidine Induced Collagen Peptide Assemblies: Morphology Modulation and Hydrolytic Catalysis Evaluation. Biomacromolecules 2018, 19 (7), 2629-2637.
53. Huang, K. Y.; Yu, C. C.; Horng, J. C. Conjugating Catalytic Polyproline Fragments with a Self-Assembling Peptide Produces Efficient Artificial Hydrolases. Biomacromolecules 2020, 21 (3), 1195-1201.
54. Agarkov, A.; Greenfield, S. J.; Ohishi, T.; Collibee, S. E.; Gilbertson, S. R. Catalysis with Phosphine-Containing Amino Acids in Various “Turn” Motifs. J. Org. Chem. 2004, 69 (23), 8077-8085.
55. Pepe, A.; Crudele, M. A.; Bochicchio, B. Effect of Proline Analogues on the Conformation of Elastin Peptides. New J. Chem. 2013, 37 (5), 1326-1335.
56. Shoulders, M. D.; Kotch, F. W.; Choudhary, A.; Guzei, I. A.; Raines, R. T. The Aberrance of the 4S Diastereomer of 4-Hydroxyproline. J. Am. Chem. Soc. 2010, 132 (31), 10857-10865.
57. Merrifield, R. B. Solid-Phase Peptide Synthesis. Adv. Enzymol. Relat. Areas Mol. Biol. 1969, 221-296.
58. Rupp, B. Circular Dichroism Spectroscopy. https://www.ruppweb.org/cd/cdtutorial.htm. (accessed on 2022/06/05)
59. Sreerama, N.; Woody, R. W., Computation and Analysis of Protein Circular Dichroism Spectra. In Methods Enzymol. 2004, 383, 318-351.
60. Kelly, S. M.; Jess, T. J.; Price, N. C. How to Study Proteins by Circular Dichroism. Biochim. Biophys. Acta 2005, 1751 (2), 119-39.
61. Wei, Y.; Thyparambil, A. A.; Latour, R. A. Protein Helical Structure Determination Using CD Spectroscopy for Solutions with Strong Background Absorbance from 190 to 230nm. Biochim. Biophys. Acta 2014, 1844 (12), 2331-7.
62. Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.; Woolfson, D. N. Installing Hydrolytic Activity into a Completely De Novo Protein Framework. Nat. Chem. 2016, 8 (9), 837-44.
63. Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. Responsive Hydrogels from the Intramolecular Folding and Self-Assembly of a Designed Peptide. J. Am.Chem. Soc. 2002, 124 (50), 15030-15037.
64. Zhang, C.; Shafi, R.; Lampel, A.; MacPherson, D.; Pappas, C. G.; Narang, V.; Wang, T.; Maldarelli, C.; Ulijn, R. V. Switchable Hydrolase Based on Reversible Formation of Supramolecular Catalytic Site Using a Self-Assembling Peptide. Angew. Chem. Int. Ed. 2017, 56 (46), 14511-14515.
65. Isom Daniel, G.; Castañeda Carlos, A.; Cannon Brian, R.; García-Moreno, E. B. Large Shifts in pKa Values of Lysine Residues Buried Inside a Protein. Proc. Natl. Acad. Sci. USA. 2011, 108 (13), 5260-5265.
66. Soto, C.; Sigurdsson, E. M.; Morelli, L.; Asok Kumar, R.; Castaño, E. M.; Frangione, B. Β-Sheet Breaker Peptides Inhibit Fibrillogenesis in a Rat Brain Model of Amyloidosis: Implications for Alzheimer's Therapy. Nat. Med. 1998, 4 (7), 822-826.
67. Moriarty, D. F.; Raleigh, D. P. Effects of Sequential Proline Substitutions on Amyloid Formation by Human Amylin20-29. Biochemistry 1999, 38 (6), 1811-1818.
68. Kanchi, P. K.; Dasmahapatra, A. K. Polyproline Chains Destabilize the Alzheimer's Amyloid-Beta Protofibrils: A Molecular Dynamics Simulation Study. J. Mol. Graph. Model. 2019, 93, 107456.
69. Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J. M.; Raussens, V. ATR-FTIR: A "Rejuvenated" Tool to Investigate Amyloid Proteins. Biochim. Biophys. Acta 2013, 1828 (10), 2328-38.
70. Hamley, I. W. Biocatalysts Based on Peptide and Peptide Conjugate Nanostructures. Biomacromolecules 2021, 22 (5), 1835-1855.
71. Luong, T. Q.; Erwin, N.; Neumann, M.; Schmidt, A.; Loos, C.; Schmidt, V.; Fandrich, M.; Winter, R. Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes. Angew. Chem. Int. Ed. 2016, 55 (40), 12412-6.
72. Friedmann, M. P.; Torbeev, V.; Zelenay, V.; Sobol, A.; Greenwald, J.; Riek, R. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening. PLOS One 2015, 10 (12), e0143948.
73. Moroz, Y. S.; Dunston, T. T.; Makhlynets, O. V.; Moroz, O. V.; Wu, Y.; Yoon, J. H.; Olsen, A. B.; McLaughlin, J. M.; Mack, K. L.; Gosavi, P. M.; van Nuland, N. A.; Korendovych, I. V. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities. J. Am. Chem. Soc. 2015, 137 (47), 14905-11.
74. Wei, Y.; Hecht, M. H. Enzyme-Like Proteins from an Unselected Library of Designed Amino Acid Sequences. Protein Eng. Des. Sel. 2004, 17 (1), 67-75.
75. Singh, N.; Conte, M. P.; Ulijn, R. V.; Miravet, J. F.; Escuder, B. Insight into the Esterase Like Activity Demonstrated by an Imidazole Appended Self-Assembling Hydrogelator. Chem. Commun. 2015, 51 (67), 13213-6.
76. Wolfenden, R. Benchmark Reaction Rates, the Stability of Biological Molecules in Water, and the Evolution of Catalytic Power in Enzymes. Annu. Rev. Biochem. 2011, 80, 645-67.
77. Yu, Z. H.; Zhang, Z. Y. Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases. Chem. Rev. 2018, 118 (3), 1069-1091.
78. Lorenz, U. Protein Tyrosine Phosphatase Assays. Curr. Proto.c Immunol. 2011, Chapter 11, Unit 11 7.
79. Der, B. S.; Edwards, D. R.; Kuhlman, B. Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering. Biochemistry 2012, 51 (18), 3933-40.
80. Wang, Y.; Yang, L.; Wang, M.; Zhang, J.; Qi, W.; Su, R.; He, Z. Bioinspired Phosphatase-Like Mimic Built from the Self-Assembly of De Novo Designed Helical Short Peptides. ACS Catal. 2021, 11 (9), 5839-5849.
81. Sarmiento, M.; Zhao, Y.; Gordon, S. J.; Zhang, Z. Y. Molecular Basis for Substrate Specificity of Protein-Tyrosine Phosphatase 1B. J. Biol. Chem. 1998, 273 (41), 26368-74.
82. O'Brien, P. J.; Herschlag, D. Alkaline Phosphatase Revisited:  Hydrolysis of Alkyl Phosphates. Biochemistry 2002, 41 (9), 3207-3225.
83. Pina, A. S.; Morgado, L.; Duncan, K. L.; Carvalho, S.; Carvalho, H. F.; Barbosa, A. J. M.; de, P. M. B.; Moreira, I. P.; Kalafatovic, D.; Morais Faustino, B. M.; Narang, V.; Wang, T.; Pappas, C. G.; Ferreira, I.; Roque, A. C. A.; Ulijn, R. V. Discovery of Phosphotyrosine-Binding Oligopeptides with Supramolecular Target Selectivity. Chem. Sci. 2021, 13 (1), 210-217.