Characterization of the Electrostatic Perturbation of a Catalytic Site (Cys)-S-/(His)-Im+H Ion-pair in One Type of Serine Proteinase Architecture by Kinetic and Computational Studies on Chemically Mutated Subtilisin Variants

Journal of Molecular Biology

Volumn 257, Issue 5, 1996, Pages 1088-1111

  Plou, Francisco J ^a,e   Kowlessur, Devanand ^a,f   Malthouse, J Paul G ^b   Mellor, Geoffrey W ^a,g   Hartshorn, Michael J ^c   Pinitglang, Surapong ^a   Patel, Hasu ^a   Topham, Christopher M ^b   Thomas, Emrys W ^d   Verma, Chandra ^c   Brocklehurst, Keith ^a  

^a QUEEN MARY UNIVERSITY OF LONDON   (United Kingdom)

^b UNIVERSITY COLLEGE DUBLIN   (Ireland)

^c UNIVERSITY OF YORK   (United Kingdom)

^d UNIVERSITY OF SALFORD   (United Kingdom)

^e INSTITUTO DE CATÁLISIS Y PETROLEOQUÍMICA   (Spain)

^f NATIONAL INSTITUTE OF MENTAL HEALTH   (United States)

^g WELLCOME RESEARCH LABORATORIES   (United Kingdom)

Author keywords

Electrostatic potential calculations; Energy minimization; Reactivity probe kinetics; Thiolate imidazolium ion pairs; Thiolsubtilisin variants

Indexed keywords

SERINE PROTEINASE; SUBTILISIN;

ARTICLE; CHEMICAL REACTION KINETICS; COMPUTER MODEL; DIELECTRIC CONSTANT; ELECTRICITY; ENZYME ACTIVE SITE; ENZYME KINETICS; ENZYME STRUCTURE; PRIORITY JOURNAL;

EID: 0029929393     PISSN: 00222836     EISSN: None     Source Type: Journal    
DOI: 10.1006/jmbi.1996.0225     Document Type: Article

References (121)

1. Abrahmsén, L., Tom, J., Burnier, J., Butcher, K. A., Kossiakoff, A. & Wells, J. A. (1991). Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution. Biochemistry, 30, 4151-4159.
2. Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971). Atomic coordinates for subtilisin BPN' (or NOVO). Biochem. Biophys. Res. Commun. 171, 337-344.
3. Allaire, M., Chernaia, M. M., Malcolm, B. A. & James, M. N. G. (1994). Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases. Nature, 369, 72-76.
4. Allewell, N. M. & Oberoi, H. (1991). Electrostatic effects in protein folding, stability, and function. Methods Enzymol. 202, 3-19.
5. Antosiewicz, J., McCammon, J. A. & Gilson, M. K. (1994). Prediction of pH-dependent properties of proteins. J. Mol. Biol., 238, 415-436.
6. a values of ionizable groups in bacteriorhodopsin. J. Mol. Biol. 224, 473-486.
7. as of ionizable groups in proteins - atomic detail from a continuum electrostatic model. Biochemistry, 29, 10219-10225.
8. Bayley, P. M., Janot, J.-M. & Martin, S. R. (1989). Subtilisin enzymes: a note on time-resolved fluorescence and circular dichroism properties. FEBS Letters, 250, 389-394.
9. 4 interactions in subtilisin 309 from Bacillus lentus. Biochemistry, 32, 2845-2852.
10. Bell, I. M., Fisher, M. L., Wu, Z.-P. & Hilvert, D. (1993). Kinetic studies on the peroxidase activity of selenosubtilisin. Biochemistry, 32, 3754-3762.
11. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., Dinola, A. & Haak, J. R. (1984). Molecular-dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684-3690.
12. Beroza, P., Fredkin, D. R., Okamura, M. Y. & Feher, G. (1991). Protonation of interacting residues in a protein by a monte carlo method: application to lysozyme and the photosynthetic reaction centre for Rhodobacter Sphaeroides. Proc. Natl Acad. Sci. USA, 88, 5804-5808.
13. Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S. & Wilson, K. (1992). Crystal structure of the alkaline proteinase savinase™ from Bacillus lentus at 1.4 Å resolution. J. Mol. Biol. 223, 427-445.
14. Bode, W., Papamokos, E., Musil, D., Seemueller, U. & Ftiz, H. (1986). Refined 1.2 Å crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin. EMBO J. 5, 813-818.
15. Bode, W., Papamokos, E. & Musil, D. (1987). The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry. Eur. J. Biochem. 166, 673-692.
16. 1′ binding sites. J. Am. Chem. Soc. 113, 1026-1030.
17. Braxton, S. & Wells, J. A. (1991). The importance of a distal hydrogen bonding group in stabilizing the transition state in subtilisin BPN'. J. Biol. Chem. 266, 11797-11800.
18. Brenner, S. (1988). The molecular evolution of genes and proteins: a tale of two serines. Nature, 334, 528-532.
19. Brocklehurst, K. (1974). The three states criterion of nucleophilic reactivity in the conjugate acid of an anionic nucleophile and some problems of proton partitioning: implications for the reactions of 2-2′-dipyridyl disulphide with papain and with L-ergothioneine. Tetrahedron, 30, 2397-2407.
20. Brocklehurst, K. (1982). Two-protonic state electrophiles as probes of enzyme mechanism. Methods Enzymol. 87C, 427-469.
21. Brocklehurst, K. (1994). A sound basis for pH-dependent kinetic studies on enzymes. Protein Eng. 7, 291-299.
22. Brocklehurst, K. & Little, G. (1972). Reactivity of the various protonic states in the reactions of papain and of L-cysteine with 2,2′- and with 4,4′-dipyridyl disulphide: evidence for nucleophilic reactivity in the un-ionized thiol group of the cysteine-25 residue of papain occasioned by its interaction with the histidine-159-asparagine-175 hydrogen-bonded system. Biochem. J. 128, 471-474.
23. Brocklehurst, K. & Malthouse, J. P. G. (1981). Evidence that the lack of high catalytic activity of thiolsubtilisin towards specific substrates may be due to an inappropriately located proton-distribution system. Demonstration of highly nucleophilic character of the thiol group of thiolsubtilisin in the catalytically relevant ionization state of the active centre by use of a two-protonic-state reactivity probe. Biochem. J. 191, 819-823.
24. Brocklehurst, S. M. & Brocklehurst, K. (1988). Analysis of pH-dependent kinetics in up to four reactive hydronic states. Biochem. J. 256, 556-558.
25. Brocklehurst, S. M., Topham, C. M. & Brocklehurst, K. (1990) A general kinetic equation for multihydronic state reactions and rapid procedures for parameter evaluation. Biochem. Soc. Trans. 18, 598-599.
26. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S. & Karplus, M. (1983). CHARMM-a program for macromolecular energy minimization, and dynamics calculations. Comput. Chem. 4, 187-217.
27. Brooks, C. L., Karplus, M. & Pettit, B. M. (1988). Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics, John Wiley & Sons, New York.
28. Brunger, A. T. & Karplus, M. (1988) Polar hydrogen positions in proteins - empirical energy placement and neutron-diffraction comparison. Proteins: Struct. Funct. Genet. 4, 148-156.
29. Carrell, R. W. & Lesk, A. M. (1994). A tale of two proteinases. The structure of two viral 3C cysteine proteinases confirm predictions of a close similarity with serine proteinases and hint at the evolutionary origins of this family of enzymes. Struct. Biol. 1, 492-494.
30. Carter, P., Abrahmsén, L. & Wells, J. A. (1991). Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'. Biochemistry, 30, 6142-6148.
31. Davis, M. E., Madura, J. D., Sines, J., Luty, B. A., Allsion, S. A. & McCammon, J. A. (1991). Diffusion-controlled enzymatic-reactions. Methods Enzymol. 202, 473-497.
32. a(s) of ionizable residues in proteins - an explicit solvent calculation for lysozyme. Proteins: Struct. Funct. Genet. 20, 85-97.
33. Dixon, W. J., Brown, M. B., Engelman, L., Hill, M. A. & Jennrich, R. I. (1988). BMDP Statistical Software, pp. 389-417, University of California Press, Guildford, UK.
34. Egmond, M. R., Antheunisse, W. P., van Bemmel, C. J., Ravestein, P., de Vlieg, J., Peters, H. & Branner, S. (1994). Engineering surface charges in a subtilisin: the effects on electrophoretic and ion-exchange behaviour. Protein Eng. 7, 793-800.
35. Elber, R. & Karplus, M. (1987). Multiple conformational states of proteins - a molecular-dynamics analysis of myoglobin. Science, 235, 318-321.
36. Gilson, M. K. (1995). Theory of electrostatic interactions in macromolecules. Curr. Opin. Struct. Biol. 5, 216-223
37. Gilson, M. K. & Honig, B. H. (1987). Calculation of electrostatic potentials in an enzyme active-site. Nature, 330, 84-86.
38. Gilson, M. K. & Honig, B. H. (1988). Energetics of charge-charge interactions in proteins. Proteins: Struct. Funct. Genet. 3, 32-52.
39. Gilson, M. K., Rashin, A., Fine, R. & Honig, B. (1985). On the calculation of electrostatic interactions in proteins. J. Mol. Biol. 184, 503-516.
40. Gilson, M. K., Sharp, K. A. & Honig, B. H. (1988). Calculating the electrostatic potential of molecules in solution: method and error assessment. J. Comput. Chem. 9, 327-335.
41. Gilson, M. K., Davis, M. E., Luty, B. A. & McCammon, J. A. (1993). Computation of electrostatic forces on solvated molecules using the Poisson-Boltzmann equation. J. Phys. Chem. 97, 3591-3600.
42. Grøn, H., Meldal, M. & Breddam, K. (1992). Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching. Biochemistry, 31, 6011-6018.
43. Guenot, J. & Kollman, P. A. (1992). Molecular-dynamics studies of a DNA-binding protein. 2. An evaluation of implicit and explicit solvent models for the molecular-dynamics simulation of the Escherichia-Coli trp repressor. Protein Sci. 1, 1185-1205.
44. Guenot, J. & Kollman, P. A. (1993). Conformational and energetic aspects of truncating non-bonded interactions in an aqueous protein dynamics simulation. J. Comput. Chem. 14, 295-311.
45. Higaki, J. N., Gibson, B. W. & Craik, C. S. (1987). Evolution of catalysis in the serine proteases. Cold Spring Harbor Symp. Quant. Biol. 52, 615-621.
46. Higaki, J. N., Evnin, L. B. & Craik, C. S. (1989). Introduction of a cysteine protease active site into trypsin. Biochemistry, 28, 9256-9263.
47. Hirono, S., Akagawa, H., Mitsui, Y. & Iitaka, Y. (1984). Crystal structure at 2.6 Å resolution of the complex of subtilisin BPN' with Streptomyces subtilisin inhibitor. J. Mol. Biol. 178, 389-413.
48. Honig, B., Sharp, K. & Yang, A. S. (1993). Macroscopic models of aqueous solutions - biological and chemical applications. J. Phys. Chem. 97, 1101-1109.
49. Honig, B. & Nicholls, A. (1995). Classical electrostatics in biology and chemistry. Science, 268, 1144-1149.
50. 77Se NMR spectroscopy. J. Am. Chem. Soc. 114, 8573-8579.
51. 1H NMR spectrostopic studies of selenosubtilisin. Biochemistry, 32, 3468-3473.
52. Ikemura, H., Takagi, H. & Inouye, M. (1987). Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem. 262, 7859-7864.
53. Jackson, S. E. & Fersht, A. R. (1993). Contribution of long-range electrostatic interactions to the stabilization of the catalytic transition state of the serine protease subtilisin BPN'. Biochemistry, 32, 13909-13916.
54. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. (1983). Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926-935.
55. JosephMcCarthy, D., Rost, L. E., Komives, E. A. & Petsko, G. A. (1994). Crystal structure of the mutant yeast triosephosphate isomerase in which the catalytic base glutamic-acid-165 is changed to aspartic-acid. Biochemistry, 33, 2824-2829.
56. Keillor, J. W., Niverov, A. A. & Brown, R. S. (1994). Catalysis of amide hydrolysis and formation under neutral conditions by a zwitterionic imidazolium thiolate. J. Am. Chem. Soc. 116, 4669-4673.
57. Klapper, I., Hagstrom, R., Fine, R., Sharp, K. & Honig, B. (1986). Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins: Struct. Funct. Genet. 1, 47-59.
58. Kowlessur, D., O'Driscoll, M., Topham, C. M., Templeton, W., Thomas, E. W. & Brocklehurst, K. (1989). The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain. Biochem. J. 259, 443-452.
59. Lewis, S. D., Johnson, F. A. & Shafer, J. A. (1976). Potentiometric determination of ionizations at the active site of papain. Biochemistry, 15, 5009-5017.
60. Li, Y. & Inouye, M. (1994). Autoprocessing of prothiolsubtilisin E in which active-dite serine 221 is altered to cysteine. J. Biol. Chem. 269, 4169-4174.
61. Linderstrøm-Lang, K. & Ottessen, M. (1947). A new protein from ovalbumin. Nature, 159, 807-808.
62. Loewenthal, R., Sancho, J., Reinikainen, T. & Fersht, A. R. (1993). Long-range surface charge-charge interactions in proteins. Comparison of experimental results with calculations from a theoretical method. J. Mol. Biol. 232, 574-583.
63. Loncharich, R. J. & Brooks, B. R. (1989). The effects of truncating long-range forces on protein dynamics. Proteins: Struct. Funct. Genet. 6, 32-45.
64. MacKerell, A. D., Sommer, M. S. & Karplus, M. (1995). pH-dependence of binding reactions from free-energy simulations and macroscopic continuum elec-trostatic calculations-applications to 2′GMP/3′GMP bonding to ribonuclease T-1 and implications for catalysis. J. Mol. Biol. 247, 774-807.
65. Madura, J. D., Briggs, J. M., Wade, R. C., Davis, M. E., Luty, B. A., Ilin, A., Antosiewicz, J., Gilson, M. K., Bagheri, B., Scott, L. R. & McCammon, J. A. (1995). Electrostatics and diffusion of molecules in solution: Simulations with the University of Houston Brownian Dynamics Program. Comput. Phys. Commun. 91, 57-95.
66. Marchwald, W., Klemm, W. & Trabert, H. (1900). Untersuchungen in der pyridinreihe II. Ber. Dtsch. Chem. Ges. 33, 1556-1566.
67. Markland, F. S. & Smith, E. L. (1971). Subtilisins: primary structure, chemical and physical properties. In The Enzymes (Boyer, R. D., ed.), 3rd edit., vol. 3, pp. 561-608, Academic Press, New York and London.
68. Matsubara, H., Kaspar, C. B., Brown, D. M. & Smith, E. L. (1965). Subtilisin BPN'. I. Physical properties and amino acid composition. J. Biol. Chem. 240, 1125-1130.
69. Matthews, D. A., Smith, W. W., Ferre, R. A., Condon, B., Budahazi, Sisson, W., Villafranca, J. E., Janson, C. A., McElroy, H. E., Gribskov, C. L. & Worland, S. (1994). Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell, 77, 761-777.
70. McPhalen, C. A. & James, M. N. G. (1988). Structural comparison of two serine proteinase-protein inhibitor complexes: Eglin-C-subtilisin Carlsberg and CI-2-subtilisin Novo. Biochemistry, 27, 6582-6598.
71. Mellor, G. W., Thomas, E. W., Topham, C. M. & Brocklehurst, K. (1993a). The ionization characteristics of the Cys-25-His-159 interactive system and of the modulatory group of papain. Resolution of ambiguity by electronic perturbation of the quasi 2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe. Biochem. J. 290, 289-296.
72. Mellor, G. W., Patel, M., Thomas, E. W. & Brocklehurst, K. (1993b). Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models. Implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu. Biochemistry, 294, 201-210.
73. Ménard, R., Khouri, H. E., Plouffe, C., Dupras, R., Ripoll, D., Vernet, T., Tessier, D. C., Laliberté, F., Thomas, D. Y. & Storer, A. C. (1990). A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain. Biochemistry, 29, 6706-6713.
74. Ménard, R., Plouffe, C., Laflamme, P., Vernet, T., Tessier, D. C., Thomas, D. Y. & Storer, A. C. (1995). Modification of the electrostatic environment is tolerated in the oxyanion hole of the cysteine proteinase papain. Biochemistry, 34, 464-471.
75. a of glu-7 and glu-75 in hen egg-white lysozyme and glu-106 in human carbonic anhydrase-II. J. Am. Chem. Soc. 113, 3572-3575.
76. Momany, F. A. & Rone, R. (1992). Validation of the general-purpose QUANTA (R) 3.2/CHARMM (R) force-field. J. Comput. Chem. 13, 888-900.
77. Neidhart, D. J. & Petsko, G. A. (1988). The refined crystal-structure of subtilisin Carlsberg at 2.5 A resolution. Protein Eng. 2, 271-276.
78. Patel, M., Thomas, M. P., Kayani, I. S., Mellor, G. W., Thomas, E. W. & Brocklehurst, K. (1993). Ficin: a cysteine proteinase with binding site-catalytic site signalling characteristics intermediate between those of papain and actinidin. Biochem. Soc. Trans. 21, 216S.
79. Philipp, M. & Bender, M. L. (1983). Kinetics of subtilisin and thiolsubtilisin. Mol. Cell Biochem. 51, 5-32.
80. Pickersgill, R. W., Sumner, I. G., Collins, M. E. & Goodenough, P. W. (1989). Structural and electrostatic differences between actinidin and papain account for differences in activity. Biochemistry, 257, 310-312.
81. Pinitglang, S., Patel, M., Thomas, E. W. & Brocklehurst, K. (1994). Existence of the Cys-His ion-pair state of cysteine proteinases may be an insufficient condition for catalytic competence. Biochem. Soc. Trans. 22, 215S.
82. Polgár, L. & Bender, M. L. (1967). The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group. Biochemistry, 6, 610-620.
83. Polgár, L. & Bender, M. L. (1969). Chromatography and activity of thiol-subtilisin. Biochemistry, 8, 136-141.
84. Polgár, L., Halász, P. & Moravcsik, E. (1973). On the reactivity of the thiol group of thiolsubtilisin. Eur. J. Biochem. 39, 421-429.
85. Rheinnecker, M., Baker, G., Eder, J. & Fersht, A. R. (1993). Engineering a novel specificity in subtilisin BPN'. Biochemistry, 32, 1199-1203.
86. Russell, A. J. & Fersht, A. R. (1986). Commercial samples of subtilisin BPN'. Nature, 321, 733.
87. Russell, A. J. & Fersht, A. R. (1987). Rational modification of enzyme catalysis by surface charge. Nature, 328, 490-500.
88. Russell, A. J., Thomas, P. G. & Fersht, A. R. (1987). Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering. J. Mol. Biol., 193, 803-813.
89. Russell, S. T. & Warshel, A. (1985). Calculations of electrostatic energies in proteins - the energetics of ionized groups in bovine pancreatic trypsin-inhibitor. J. Mol. Biol. 185, 389-404.
90. Salih, E., Malthouse, J. P. G., Kowlessur, D., Jarvis, M., O'Driscoll, M. & Brocklehurst, K. (1987). Differences in the chemical and catalytic characteristics of two crystallographically "identical" enzyme catalytic sites. Characterisation of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment. Biochem. J. 247, 181-193.
91. Shih, H. H. L., Brady, J. & Karplus, M. (1985). Structure of proteins with single-site mutations - a minimum perturbation approach. Proc. Natl Acad. Sci. USA, 82, 1697-1700.
92. Shipton, M. & Brocklehurst, K. (1978). Characterization of the papain active centre using two-protonic-state-electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system. Biochem. J. 171, 385-401.
93. I of the essential histidine-cysteine interactive systems of papain, bromelain and ficin. FEBS Letters, 50, 365-368.
94. Smith, P. E., Brunne, R. M., Mark, A. E. & van Gunsteren, W. F. (1993). Dielectric properties of trypsin-inhibitor and lysozyme calculated from molecular-dynamics simulations. J. Phys. Chem. 97, 2009-2014.
95. Soman, K., Yang, A. S., Honig, B. & Fletterick, R. (1989). Electrical potentials in trypsin inhibitor. Biochemistry, 28, 9918-9926.
96. Sternberg, M. J. E., Hayes, F. R. F., Russell, A. J., Thomas, P. G. & Fersht, A. R. (1987). Prediction of electrostatic effects of engineering of protein charges. Nature, 330, 86-88.
97. Strausberg, S., Alexander, P., Wang, L., Schwarz, F. & Bryan, P. (1993). Catalysis of a protein folding reaction: Thermodynamic and kinetic analysis of subtilisin BPN' interactions with its propeptide fragment. Biochemistry, 32, 8112-8119.
98. 2O by imidazole to thiol esters as a model for the deacylation step. J. Am. Chem. Soc. 107, 7669-7679.
99. Stuchbury, T., Shipton, M., Norris, R., Malthouse, J. P. G., Brocklehurst, K., Herbert, J. A. L. & Suschitzky, H. (1975). A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem. J. 151, 417-432.
100. Svendsen, I. B. (1976). Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review. Carlsberg Res. Commun. 41, 238-291.
101. Syed, R., Wu, Z.-P., Hogle, J. M. & Hilvert, D. (1993). Crystal structure of selenosubtilisin at 2.0-Å resolution. Biochemistry, 32, 6157-6164.
102. Takeuchi, Y., Noguchi, S., Satow, Y., Kojima, S., Kumagai, I., Miura, K.-i., Nakamura, K. T. & Mitsui, Y (1991a). Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor). Protein Eng. 4, 501-508.
103. Takeuchi, Y., Satow, Y., Nakamura, K. T. & Mitsui, Y. (1991b) Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution. J. Mol. Biol. 221, 309-325.
104. Tanner, J. J., Smith, P. E. & Krause, K. L. (1993). Molecular-dynamics simulations and rigid-body (TLS) analysis of aspartate carbamoyltransferase -evidence for an uncoupled R-state. Protein Sci. 2, 927-935.
105. Taylor, M. A. J., Baker, K. C., Connerton, I. R., Cummings, N. J., Harris, G. W., Henderson, I. M. J., Jones, S. T., Pickersgill, R. W., Sumner, I. G., Warwicker, J. & Goodenough, P. W. (1994). An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions. Protein Eng. 7, 1267-1276.
106. Thomas, M. P., Topham, C. M., Kowlessur, D., Mellor, G. W., Thomas, E. W., Whitford, D. & Brocklehurst, K. (1994). Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active centre characteristics determined by pH-dependent kinetics of catalysis and of reactions with time-dependent inhibitors: the Cys25/His-159 ion pair is insufficient for catalytic competence in both chymopapain M and papain. Biochem. J. 300, 805-820.
107. Thomas, M. P., Verma, C., Boyd, S. M. & Brocklehurst, K. (1995). The structural origins of the unusual specificities observed in the isolation of chymopapain-M and actinidin by covalent chromatography and the lack of inhibition of chymopapain-M by cystatin. Biochem. J. 306, 39-46.
108. Topham, C. M., Salih, E., Frazao, C., Kowlessur, D., Overington, J. P., Thomas, M., Brocklehurst, S. M., Patel, M., Thomas, E. W. & Brocklehurst, K. (1991). Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase Ω deduced by knowledge-based modelling and active centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis. Biochem. J. 280, 79-92.
109. van Gunsteren, W. F. & Karplus, M. (1982). Effect of constraints on the dynamics of macromolecules. Macromolecules, 15, 1528-1544.
110. Wang, J., Xiang, X. F. & Lim, C. (1994). The double catalytic triad, Cys250-His159-Asp158 and Cys25-His159-Asn175, in papain catalysis - role of Asp158 and Asn175. Protein Eng. 7, 75-82.
111. Warshel, A., Sussman, F. & King, G. (1986). Free-energy of charges in solvated poteins - microscopic calculations using a reversible charging process. Biochemistry, 25, 8368-8372.
112. Warwicker, J. (1994). Improved continuum electrostatic modelling in proteins, with comparison to experiment. J. Mol. Biol. 236, 887-903.
113. Wells, J. A., Cunningham, B. C. Graycar, T. P. & Estell, D. A. (1987). Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering. Proc. Natl Acad. Sci. USA, 84, 5167-5171.
114. Wilke, M. E., Higaki, J. N., Craik, C. S. & Fletterick, R. J. (1991). Crystal structure of rat trypsin-S195C at -150°C. Analysis of low activity of recombinant and semisynthetic thiol proteases. J. Mol. Biol. 219, 511-523.
115. Willenbrock, F. & Brocklehurst, K. (1984). Natural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-Dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state reactivity probe (2,2′-dipyridyl disulphide) and with a specific synthetic substrate (N-benzyloxy-carbonyl-L-arginyl-L-arginine-2-naphthylamide). Biochem. J. 222, 805-814.
116. Willenbrock, F. & Brocklehurst, K. (1985). A general framework for cysteine proteinase mechanism from studies on enzymes with structurally different analogous catalytic site residues Asp 158 & 161 (papain and actinidin), Gly 196 (cathepsin B) and Asn 165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-α-benzyloxy-carbonyl-L-arginyl-L-arginine-2-naphthylamide catalysed by cathepsin B and of L-arginine-2-naphthylamide catalysed by cathepsin H. Biochem. J. 227, 521-528.
117. Willenbrock, F., Knight, C. G., Murphy, G., Phillips, I. R. & Brocklehurst, K. (1995). Evidence for the importance of weakly bound water for matrix metalloproteinase activity. Biochemistry, 34, 12012-12018.
118. Wu, Z.-P. & Hilvert, D. (1989). Conversion of a protease into an acyl transferase: selenolsubtilisin. J. Am. Chem. Soc. 111, 4513-4514.
119. Yang, A. S. & Honig, B. (1993). On the pH-dependence of protein stability. J. Mol. Biol. 231, 459-474.
120. a(s) in proteins. Proteins: Struct. Funct. Genet. 15, 252-265.
121. Yokosawa, H., Ojima, S. & Ishii, S. (1977). Thioltrypsin: chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue. J. Biochem. 82, 869-876.