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Unexpected crucial role of residue 225 in serine proteases.

Guinto ER, Caccia S, Rose T, Fütterer K, Waksman G, Di Cera E
Proc Natl Acad Sci U S A 96 1852-7 (1999)
Related entries: 1thp, 2thf

Cited: 49 times
EuropePMC logo PMID: 10051558

Abstract

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Articles - 1b7x mentioned but not cited (2)

  1. Crystal structure of an RNA aptamer bound to thrombin. Long SB, Long MB, White RR, Sullenger BA. RNA 14 2504-2512 (2008)
  2. Unexpected crucial role of residue 225 in serine proteases. Guinto ER, Caccia S, Rose T, Fütterer K, Waksman G, Di Cera E. Proc Natl Acad Sci U S A 96 1852-1857 (1999)


Reviews citing this publication (6)

  1. Role of Na+ and K+ in enzyme function. Page MJ, Di Cera E. Physiol Rev 86 1049-1092 (2006)
  2. Thrombin. Di Cera E. Mol Aspects Med 29 203-254 (2008)
  3. Thrombin domains: structure, function and interaction with platelet receptors. De Cristofaro R, De Candia E. J Thromb Thrombolysis 15 151-163 (2003)
  4. Molecular modeling and biocatalysis: explanations, predictions, limitations, and opportunities. Kazlauskas RJ. Curr Opin Chem Biol 4 81-88 (2000)
  5. Thrombin allostery. Di Cera E, Page MJ, Bah A, Bush-Pelc LA, Garvey LC. Phys Chem Chem Phys 9 1291-1306 (2007)
  6. Thrombin: a paradigm for enzymes allosterically activated by monovalent cations. Di Cera E. C R Biol 327 1065-1076 (2004)

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  7. Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle. Gaboriaud C, Rossi V, Bally I, Arlaud GJ, Fontecilla-Camps JC. EMBO J 19 1755-1765 (2000)
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  13. Structure-based predictive models for allosteric hot spots. Demerdash ON, Daily MD, Mitchell JC. PLoS Comput Biol 5 e1000531 (2009)
  14. Engineering thrombin for selective specificity toward protein C and PAR1. Marino F, Pelc LA, Vogt A, Gandhi PS, Di Cera E. J Biol Chem 285 19145-19152 (2010)
  15. Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding. Schmidt AE, Stewart JE, Mathur A, Krishnaswamy S, Bajaj SP. J Mol Biol 350 78-91 (2005)
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  17. Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold. Vogt AD, Chakraborty P, Di Cera E. J Biol Chem 290 22435-22445 (2015)
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  19. Thermodynamics of Na+ binding to coagulation serine proteases. Griffon N, Di Stasio E. Biophys Chem 90 89-96 (2001)
  20. Computationally-driven identification of antibody epitopes. Hua CK, Gacerez AT, Sentman CL, Ackerman ME, Choi Y, Bailey-Kellogg C. Elife 6 e29023 (2017)
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  22. Mutant N143P reveals how Na+ activates thrombin. Niu W, Chen Z, Bush-Pelc LA, Bah A, Gandhi PS, Di Cera E. J Biol Chem 284 36175-36185 (2009)
  23. Prostate-specific antigen is a "chymotrypsin-like" serine protease with unique P1 substrate specificity. LeBeau AM, Singh P, Isaacs JT, Denmeade SR. Biochemistry 48 3490-3496 (2009)
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  25. High resolution crystal structures of free thrombin in the presence of K(+) reveal the molecular basis of monovalent cation selectivity and an inactive slow form. Carrell CJ, Bush LA, Mathews FS, Di Cera E. Biophys Chem 121 177-184 (2006)
  26. Molecular cloning of novel serine proteases and phospholipases A2 from green pit viper (Trimeresurus albolabris) venom gland cDNA library. Rojnuckarin P, Muanpasitporn C, Chanhome L, Arpijuntarangkoon J, Intragumtornchai T. Toxicon 47 279-287 (2006)
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  29. Expression, purification and characterization of factor IX derivatives using a novel vector system. Yang L, Gopalakrishna K, Manithody C, Rezaie AR. Protein Expr Purif 50 196-202 (2006)
  30. Role of the I16-D194 ionic interaction in the trypsin fold. Stojanovski BM, Chen Z, Koester SK, Pelc LA, Di Cera E. Sci Rep 9 18035 (2019)
  31. Sodium-site in serine protease domain of human coagulation factor IXa: evidence from the crystal structure and molecular dynamics simulations study. Vadivel K, Schreuder HA, Liesum A, Schmidt AE, Goldsmith G, Bajaj SP. J Thromb Haemost 17 574-584 (2019)
  32. Murine serine proteases MASP-1 and MASP-3, components of the lectin pathway activation complex of complement, are encoded by a single structural gene. Stover CM, Lynch NJ, Dahl MR, Hanson S, Takahashi M, Frankenberger M, Ziegler-Heitbrock L, Eperon I, Thiel S, Schwaeble WJ. Genes Immun 4 374-384 (2003)
  33. WEDGE: an anticoagulant thrombin mutant produced by autoactivation. Wood DC, Pelc LA, Pozzi N, Wallisch M, Verbout NG, Tucker EI, Gruber A, Di Cera E. J Thromb Haemost 13 111-114 (2015)
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  35. Expression and functional characterization of chitribrisin, a thrombin-like enzyme, in the venom of the Chinese green pit viper (Trimeresurus albolabris). Lin Y, Yu X, He Q, Li H, Li D, Song X, Wang Y, Wen H, Deng H, Deng J. Protein Expr Purif 67 48-52 (2009)
  36. Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex. Yang L, Manithody C, Qureshi SH, Rezaie AR. Biochemistry 47 5976-5985 (2008)
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  38. Acidosis, magnesium and acetylsalicylic acid: effects on thrombin. Borisevich N, Loznikova S, Sukhodola A, Halets I, Bryszewska M, Shcharbin D. Spectrochim Acta A Mol Biomol Spectrosc 104 158-164 (2013)
  39. Bi-factor analysis based on noise-reduction (BIFANR): a new algorithm for detecting coevolving amino acid sites in proteins. Liu J, Duan X, Sun J, Yin Y, Li G, Wang L, Liu B. PLoS One 8 e79764 (2013)
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  41. Complement component factor B has thrombin-like activity. Takahashi K, Banda NK, Holers VM, Van Cott EM. Biochem Biophys Res Commun 552 17-22 (2021)


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