Abstract
Serine proteinase inhibitor Kazal-type 2 (SPINK2), a human seminal plasma proteinase inhibitor, is defined by a Kazal domain, which contains six conserved cysteine residues and forms a disulfide bridge-stabilized conformation. It has been recognized that SPINK2 is a trypsin/acrosin inhibitor involving in fertilization, however, only a few studies focused on SPINK2 during past decades. Recently, the SPINK2 gene was reported to show the greatest differential expression levels in primary cutaneous follicle center cell lymphomas (PCFCCLs). To elucidate the molecular functions of SPINK2 in non-germ cells, quantitative real-time PCR was employed by our collaborator to confirm the expression profile of SPINK2 in several leukemia cells and the invasion assays were also performed to verify the SPINK2 gene was a metastasis-enhancer in lung cancer cells. We here determined the binding site for trypsin by site-directed mutagenesis approach and the three-dimensional solution structure by nuclear magnetic resonance (NMR) spectroscopy. By using an effective strategy, recombinant SPINK proteins were successfully obtained from Escherichia coli and further employed in structural and functional analyses. By using trypsin activity assay, the results showed that SPINK2 has an ability to inhibit trypsin and the trypsin-binding region, P2-P2□ site (Pro23-Arg24-His25-Phe26), was defined. Furthermore, eight point mutations within this region (P23A, P23S, P23T, R24A, H25A, H25I, H25E and F26A) were produced to determine the influences of trypsin-inhibitory activities on each site. Enzymatic kinetic studies were also carried out to calculate the inhibition constants Ki for trypsin. NMR spectroscopic results revealed that SPINK2 comprises an α-helix and one triple-stranded anti-parallel β-sheet stabilized by three disulfide bonds, which is similar to pancreatic secretory trypsin inhibitors (PSTI or SPINK1). The putative trypsin-SPINK2 complex structure was predicted by computational docking analysis. Prediction of the binding site between trypsin and SPINK2 is consistent with the results of experimental mutagenesis analysis. These studies not only provide critical information about the structural data and fundamentally biophysical features on SPINK2, but also suggest its possible effect on tumor progression and metastasis.

Contents
Abstract in Chinese………………………………………………..…..2
Abstract……………………………………………………………..….4
Abbreviations………………………………………………………..…6

Chapter I. Introduction
1.1 Serine proteinase…………………………………………………..…….8
1.2 Serine proteinase inhibitor, Kazal type (SPINK)………………….........10
1.3 The reactive sites of trypsin inhibitors………………………………….14
1.4 The purpose of this thesis……………………………………………….15
1.5 Figures…………………………………………………………………..16

Chapter II. Experimental Procedures
2.1 Construction of Plasmids………………………………………………..22
2.2 Production and Purification of Recombinant SPINK Proteins………….24
2.3 Circular Dichroism (CD) Experiments………………………………….25
2.4 Serine Proteinases Inhibition Assays………………………...…...……..26
2.5 Enzyme Kinetic Assay………………………………………….……….28
2.6 NMR Experiments and Structure Calculations…………………………29
2.7 Molecular Docking……………………………………………….……..31
2.8 Figures……………………………………………………………..……32

Chapter III. Results & Discussions
3.1 Production and Purification of Recombinant SPINK Proteins……....…45
3.2 Determination of Secondary Structure of SPINK and Mutants……..….46
3.3 Inhibiting assays of several serine proteinases……………………….…47
3.4 Reactive Site of SPINK2 for Trypsin Inhibition………………………..49
3.5 Determination of the Inhibition Constant of SPINK2 for Trypsin……...51
3.6 Resonance Assignment and Molecular Structure…………………….…51
3.7 Docking Analysis of the Trypsin-SPINK2 Complex……………….…..54
3.8 Figures and Tables………………………………………………………57

Chapter IV. Conclusions…………………………………….………...89

References………………………………………………………………95

References:
(1) Jackson, R. C., and Blobel, G. (1977) Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A 74, 5598-602.
(2) Ichihara, S., Beppu, N., and Mizushima, S. (1984) Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity. J Biol Chem 259, 9853-7.
(3) Davie, E. W., and Fujikawa, K. (1977) The role of serine proteinases in the early phase of blood coagulation [proceedings]. Biochem Soc Trans 5, 1241-3.
(4) Cesarman-Maus, G., and Hajjar, K. A. (2005) Molecular mechanisms of fibrinolysis. Br J Haematol 129, 307-21.
(5) Pillai, S., Botti, R., Jr., and Zull, J. E. (1983) ATP activation of parathyroid hormone cleavage catalyzed by cathepsin D from bovine kidney. J Biol Chem 258, 9724-8.
(6) Tyndall, J. D., Nall, T., and Fairlie, D. P. (2005) Proteases universally recognize beta strands in their active sites. Chem Rev 105, 973-99.
(7) Hedstrom, L. (2002) Serine protease mechanism and specificity. Chem Rev 102, 4501-24.
(8) Rawlings, N. D., Morton, F. R., Kok, C. Y., Kong, J., and Barrett, A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res 36, D320-5.
(9) Koivunen, E., Huhtala, M. L., and Stenman, U. H. (1989) Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase. J Biol Chem 264, 14095-9.
(10) Koshikawa, N., Nakamura, T., Tsuchiya, N., Isaji, M., Yasumitsu, H., Umeda, M., and Miyazaki, K. (1996) Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells. J Biochem (Tokyo) 119, 334-9.
(11) Sorsa, T., Salo, T., Koivunen, E., Tyynela, J., Konttinen, Y. T., Bergmann, U., Tuuttila, A., Niemi, E., Teronen, O., Heikkila, P., Tschesche, H., Leinonen, J., Osman, S., and Stenman, U. H. (1997) Activation of type IV procollagenases by human tumor-associated trypsin-2. J Biol Chem 272, 21067-74.
(12) Miyata, S., Miyagi, Y., Koshikawa, N., Nagashima, Y., Kato, Y., Yasumitsu, H., Hirahara, F., Misugi, K., and Miyazaki, K. (1998) Stimulation of cellular growth and adhesion to fibronectin and vitronectin in culture and tumorigenicity in nude mice by overexpression of trypsinogen in human gastric cancer cells. Clin Exp Metastasis 16, 613-22.
(13) Nyberg, P., Moilanen, M., Paju, A., Sarin, A., Stenman, U. H., Sorsa, T., and Salo, T. (2002) MMP-9 activation by tumor trypsin-2 enhances in vivo invasion of human tongue carcinoma cells. J Dent Res 81, 831-5.
(14) Stenman, U. H. (2002) Tumor-associated trypsin inhibitor. Clin Chem 48, 1206-9.
(15) Stenman, U. H., Koivunen, E., and Vuento, M. (1988) Characterization of a tumor-associated serine protease. Biol Chem Hoppe Seyler 369 Suppl, 9-14.
(16) Moilanen, M., Sorsa, T., Stenman, M., Nyberg, P., Lindy, O., Vesterinen, J., Paju, A., Konttinen, Y. T., Stenman, U. H., and Salo, T. (2003) Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen. Biochemistry 42, 5414-20.
(17) Emi, M., Nakamura, Y., Ogawa, M., Yamamoto, T., Nishide, T., Mori, T., and Matsubara, K. (1986) Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41, 305-10.
(18) Tani, T., Kawashima, I., Mita, K., and Takiguchi, Y. (1990) Nucleotide sequence of the human pancreatic trypsinogen III cDNA. Nucleic Acids Res 18, 1631.
(19) Wiegand, U., Corbach, S., Minn, A., Kang, J., and Muller-Hill, B. (1993) Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136, 167-75.
(20) Koshikawa, N., Yasumitsu, H., Umeda, M., and Miyazaki, K. (1992) Multiple secretion of matrix serine proteinases by human gastric carcinoma cell lines. Cancer Res 52, 5046-53.
(21) Laskowski, M., Jr., and Kato, I. (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49, 593-626.
(22) Silverman, G. A., Bird, P. I., Carrell, R. W., Church, F. C., Coughlin, P. B., Gettins, P. G., Irving, J. A., Lomas, D. A., Luke, C. J., Moyer, R. W., Pemberton, P. A., Remold-O'Donnell, E., Salvesen, G. S., Travis, J., and Whisstock, J. C. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276, 33293-6.
(23) Irving, J. A., Pike, R. N., Lesk, A. M., and Whisstock, J. C. (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 10, 1845-64.
(24) Irving, J. A., Steenbakkers, P. J., Lesk, A. M., Op den Camp, H. J., Pike, R. N., and Whisstock, J. C. (2002) Serpins in prokaryotes. Mol Biol Evol 19, 1881-90.
(25) Luckett, S., Garcia, R. S., Barker, J. J., Konarev, A. V., Shewry, P. R., Clarke, A. R., and Brady, R. L. (1999) High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds. J Mol Biol 290, 525-33.
(26) Loebermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J Mol Biol 177, 531-57.
(27) Clynen, E., Schoofs, L. and Salzet, M. (2005) A review of the most important classes of serine protease inhibitors in insects and leeches. Medicinal Chemistry Reviews 2, 197-206.
(28) Laskowski, M., Jr. (1986) Protein inhibitors of serine proteinases--mechanism and classification. Adv Exp Med Biol 199, 1-17.
(29) Bode, W., and Huber, R. (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204, 433-51.
(30) Rawlings, N. D., Tolle, D. P., and Barrett, A. J. (2004) Evolutionary families of peptidase inhibitors. Biochem J 378, 705-16.
(31) Kazal, L., Spicer, D., and Brahinsky, R. (1948) Isolation of a crystalline trypsin inhibitor-anticoagulant protein from pancreas. J Am Chem Soc 79, 3034-40.
(32) Wapenaar, M. C., Monsuur, A. J., Poell, J., van 't Slot, R., Meijer, J. W., Meijer, G. A., Mulder, C. J., Mearin, M. L., and Wijmenga, C. (2007) The SPINK gene family and celiac disease susceptibility. Immunogenetics 59, 349-57.
(33) Rinderknecht, H. (1993) Pancreatic secretory enzymes. In: Go VWL, DiMagno EP; Gardner JD, et al, eds, New York: Raven.
(34) Stenman, U. H., Koivunen, E., Itkonen, O., and Halila, H. (1993) Clinical use and biological function of tumor-associated trypsin inhibitor (TATI). Torino: Edizioni Minerva Medica S.P.A., 351-61.
(35) Lukkonen, A., Lintula, S., von Boguslawski, K., Carpen, O., Ljungberg, B., Landberg, G., and Stenman, U. H. (1999) Tumor-associated trypsin inhibitor in normal and malignant renal tissue and in serum of renal-cell carcinoma patients. Int J Cancer 83, 486-90.
(36) Solakidi, S., Dessypris, A., Stathopoulos, G. P., Androulakis, G., and Sekeris, C. E. (2004) Tumour-associated trypsin inhibitor, carcinoembryonic antigen and acute-phase reactant proteins CRP and alpha1-antitrypsin in patients with gastrointestinal malignancies. Clin Biochem 37, 56-60.
(37) Lohr, J. M. (2007) What are the useful biological and functional markers of early-stage chronic pancreatitis? J Gastroenterol 42 Suppl 17, 66-71.
(38) Wiksten, J. P., Lundin, J., Nordling, S., Kokkola, A., Stenman, U. H., and Haglund, C. (2005) High tissue expression of tumour-associated trypsin inhibitor (TATI) associates with a more favourable prognosis in gastric cancer. Histopathology 46, 380-8.
(39) Dietl, T., Kruck, J., Schill, W. B., and Fritz, H. (1976) Localization of seminal plasma proteinase inhibitors in human spermatozoa as revealed by the indirect immunofluorescence technique. Hoppe Seylers Z Physiol Chem 357, 1333-7.
(40) Fink, E., Hehlein-Fink, C., and Eulitz, M. (1990) Amino acid sequence elucidation of human acrosin-trypsin inhibitor (HUSI-II) reveals that Kazal-type proteinase inhibitors are structurally related to beta-subunits of glycoprotein hormones. FEBS Lett 270, 222-4.
(41) Moritz, A., Lilja, H., and Fink, E. (1991) Molecular cloning and sequence analysis of the cDNA encoding the human acrosin-trypsin inhibitor (HUSI-II). FEBS Lett 278, 127-30.
(42) Rockett, J. C., Patrizio, P., Schmid, J. E., Hecht, N. B., and Dix, D. J. (2004) Gene expression patterns associated with infertility in humans and rodent models. Mutat Res 549, 225-40.
(43) Hoefnagel, J. J., Dijkman, R., Basso, K., Jansen, P. M., Hallermann, C., Willemze, R., Tensen, C. P., and Vermeer, M. H. (2005) Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling. Blood 105, 3671-8.
(44) He, X., Gonzalez, V., Tsang, A., Thompson, J., Tsang, T. C., and Harris, D. T. (2005) Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells. Stem Cells Dev 14, 188-98.
(45) Norgett, E. E., and Kelsell, D. P. (2002) SPINK5: both rare and common skin disease. Trends Mol Med 8, 7.
(46) Bitoun, E., Chavanas, S., Irvine, A. D., Lonie, L., Bodemer, C., Paradisi, M., Hamel-Teillac, D., Ansai, S., Mitsuhashi, Y., Taieb, A., de Prost, Y., Zambruno, G., Harper, J. I., and Hovnanian, A. (2002) Netherton syndrome: disease expression and spectrum of SPINK5 mutations in 21 families. J Invest Dermatol 118, 352-61.
(47) Schechter, I., and Berger, A. (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27, 157-62.
(48) Otlewski, J., Jaskolski, M., Buczek, O., Cierpicki, T., Czapinska, H., Krowarsch, D., Smalas, A. O., Stachowiak, D., Szpineta, A., and Dadlez, M. (2001) Structure-function relationship of serine protease-protein inhibitor interaction. Acta Biochim Pol 48, 419-28.
(49) Schlott, B., Wohnert, J., Icke, C., Hartmann, M., Ramachandran, R., Guhrs, K. H., Glusa, E., Flemming, J., Gorlach, M., Grosse, F., and Ohlenschlager, O. (2002) Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies. J Mol Biol 318, 533-46.
(50) Kamei, K., Sato, S., Hamato, N., Takano, R., Ohshima, K., Yamamoto, R., Nishino, T., Kato, H., and Hara, S. (2000) Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases. Biochim Biophys Acta 1480, 6-12.
(51) Battiste, J. L., Li, R., and Woodward, C. (2002) A highly destabilizing mutation, G37A, of the bovine pancreatic trypsin inhibitor retains the average native conformation but greatly increases local flexibility. Biochemistry 41, 2237-45.
(52) Castro, M. J., and Anderson, S. (1996) Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin. Biochemistry 35, 11435-46.
(53) Khamrui, S., Dasgupta, J., Dattagupta, J. K., and Sen, U. (2005) Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 Å) and biochemical basis. Biochim Biophys Acta 1752, 65-72.
(54) Grzesiak, A., Helland, R., Smalas, A. O., Krowarsch, D., Dadlez, M., and Otlewski, J. (2000) Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases. J Mol Biol 301, 205-17.
(55) Lin, K. F., Lee, T. R., Tsai, P. H., Hsu, M. P., Chen, C. S., and Lyu, P. C. (2007) Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin. Proteins 68, 530-40.
(56) Xiong, A. S., Yao, Q. H., Peng, R. H., Li, X., Fan, H. Q., Cheng, Z. M., and Li, Y. (2004) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32, e98.
(57) Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1995) Short protocols in molecular biology, 3 ed., John Wiley & Sons, Boston.
(58) Landon, M. (1977) Cleavage at aspartyl-prolyl bonds. Methods Enzymol 47, 145-9.
(59) Elmorjani, K., Lurquin, V., Lelion, A., Rogniaux, H., and Marion, D. (2004) A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins. Biochem Biophys Res Commun 316, 1202-9.
(60) Greenfield, N., and Fasman, G. D. (1969) Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4108-16.
(61) Johnson, W. C., Jr. (1990) Protein secondary structure and circular dichroism: a practical guide. Proteins 7, 205-14.
(62) Winnica, D. E., Novella, M. L., Dematteis, A., and Coronel, C. E. (2000) Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies. Biol Reprod 63, 42-8.
(63) Somprasong, N., Rimphanitchayakit, V., and Tassanakajon, A. (2006) A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. Dev Comp Immunol 30, 998-1008.
(64) Samuel, D., Liu, Y. J., Cheng, C. S., and Lyu, P. C. (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa). J Biol Chem 277, 35267-73.
(65) Cheng, C. S., Samuel, D., Liu, Y. J., Shyu, J. C., Lai, S. M., Lin, K. F., and Lyu, P. C. (2004) Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense. Biochemistry 43, 13628-36.
(66) Lin, K. F., Liu, Y. N., Hsu, S. T., Samuel, D., Cheng, C. S., Bonvin, A. M., and Lyu, P. C. (2005) Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean. Biochemistry 44, 5703-12.
(67) Liu, Y. J., Cheng, C. S., Lai, S. M., Hsu, M. P., Chen, C. S., and Lyu, P. C. (2006) Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins 63, 777-86.
(68) Liu, Y. N., Lai, Y. T., Chou, W. I., Chang, M. D., and Lyu, P. C. (2007) Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase. Biochem J 403, 21-30.
(69) Grzesiek, S., and Bax, A. (1993) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR 3, 185-204.
(70) Brunger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Crystallography & NMR System. Acta Cryst. D54, 905-921.
(71) Brutscher, B. (2002) Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment. J Magn Reson 156, 155-9.
(72) Permi, P. (2002) Intraresidual HNCA: an experiment for correlating only intraresidual backbone resonances. J Biomol NMR 23, 201-9.
(73) Kupce, E., Muhandiram, D. R., and Kay, L. E. (2003) A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance. J Biomol NMR 27, 175-9.
(74) Goddard, T. D., and Kneller, D. G. (1999), University of California, San Francisco.
(75) Linge, J. P., Williams, M. A., Spronk, C. A., Bonvin, A. M., and Nilges, M. (2003) Refinement of protein structures in explicit solvent. Proteins 50, 496-506.
(76) Nederveen, A. J., Doreleijers, J. F., Vranken, W., Miller, Z., Spronk, C. A., Nabuurs, S. B., Guntert, P., Livny, M., Markley, J. L., Nilges, M., Ulrich, E. L., Kaptein, R., and Bonvin, A. M. (2005) RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank. Proteins 59, 662-72.
(77) Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291.
(78) Vakser, I. A., Matar, O. G., and Lam, C. F. (1999) A systematic study of low-resolution recognition in protein--protein complexes. Proc Natl Acad Sci U S A 96, 8477-82.
(79) Berendsen, H. J. C., van Der Spoel, D., and van Drunen, R. (1995) ROMACS: A message-passing parallel molecular dynamics implementation Comp. Phys. Comm. 91, 43-56.
(80) Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A. E., and Berendsen, H. J. (2005) GROMACS: fast, flexible, and free. J Comput Chem 26, 1701-18.
(81) Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8, 127-34.
(82) Wuthrich, K. (1986) NMR of Proteins and nucleic acids, John Wiley & Sons, New York.
(83) Bode, W., and Schwager, P. (1975) The refined crystal structure of bovine beta-trypsin at 1.8 Å resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. J Mol Biol 98, 693-717.
(84) Bode, W., Schwager, P., and Huber, R. (1978) The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 Å resolution. J Mol Biol 118, 99-112.
(85) Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-80.
(86) Koradi, R., Billeter, M., and Wuthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14, 51-5, 29-32.
(87) Delano, W. L. (2002) The PyMOL Molecular Graphics System., Delano Scientific, San Carlos, CA, USA.
(88) DiscoveryStudio. (2002), Accelrys Inc., San Diego, CA.
(89) Maywald, F., Boldicke, T., Gross, G., Frank, R., Blocker, H., Meyerhans, A., Schwellnus, K., Ebbers, J., Bruns, W., Reinhardt, G., and et al. (1988) Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli. Gene 68, 357-69.
(90) Hess, G. P., McConn, J., Ku, E., and McConkey, G. (1970) Studies of the activity of chymotrypsin. Philos Trans R Soc Lond B Biol Sci 257, 89-104.
(91) Kraut, J. (1977) Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem 46, 331-58.
(92) Steitz, T. A., and Shulman, R. G. (1982) Crystallographic and NMR studies of the serine proteases. Annu Rev Biophys Bioeng 11, 419-44.
(93) Ash, E. L., Sudmeier, J. L., Day, R. M., Vincent, M., Torchilin, E. V., Haddad, K. C., Bradshaw, E. M., Sanford, D. G., and Bachovchin, W. W. (2000) Unusual 1H NMR chemical shifts support (His) C(epsilon) 1...O==C H-bond: proposal for reaction-driven ring flip mechanism in serine protease catalysis. Proc Natl Acad Sci U S A 97, 10371-6.