1gbt › Citations

Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.

Mangel WF, Singer PT, Cyr DM, Umland TC, Toledo DL, Stroud RM, Pflugrath JW, Sweet RM
Biochemistry 29 8351-7 (1990)
Cited: 22 times
EuropePMC logo PMID: 2252895

Abstract

The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Articles - 1gbt mentioned but not cited (3)

  1. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Radisky ES, Lee JM, Lu CJ, Koshland DE. Proc. Natl. Acad. Sci. U.S.A. 103 6835-6840 (2006)
  2. Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1. Brucz K, Miknis ZJ, Schultz LW, Umland TC. Protein Expr. Purif. 52 249-257 (2007)
  3. Molecular mechanism of inhibiting the SARS-CoV-2 cell entry facilitator TMPRSS2 with camostat and nafamostat. Hempel T, Raich L, Olsson S, Azouz NP, Klingler AM, Hoffmann M, Pöhlmann S, Rothenberg ME, Noé F. Chem Sci 12 983-992 (2021)


Reviews citing this publication (3)

  1. Understanding enzymic catalysis: the importance of short, strong hydrogen bonds. Gerlt JA, Kreevoy MM, Cleland W, Frey PA. Chem. Biol. 4 259-267 (1997)
  2. Divining the serpin inhibition mechanism: a suicide substrate 'springe'? Engh RA, Huber R, Bode W, Schulze AJ. Trends Biotechnol. 13 503-510 (1995)
  3. Time-resolved protein crystallography. Johnson LN. Protein Sci. 1 1237-1243 (1992)

Articles citing this publication (16)

  1. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Ren D, Pipes GD, Liu D, Shih LY, Nichols AC, Treuheit MJ, Brems DN, Bondarenko PV. Anal. Biochem. 392 12-21 (2009)
  2. Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase. Ponnuraj K, Xu Y, Macon K, Moore D, Volanakis JE, Narayana SV. Mol. Cell 14 17-28 (2004)
  3. Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction. Finer-Moore JS, Kossiakoff AA, Hurley JH, Earnest T, Stroud RM. Proteins 12 203-222 (1992)
  4. Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity. Jing H, Babu YS, Moore D, Kilpatrick JM, Liu XY, Volanakis JE, Narayana SV. J. Mol. Biol. 282 1061-1081 (1998)
  5. Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets. Katz BA, Sprengeler PA, Luong C, Verner E, Elrod K, Kirtley M, Janc J, Spencer JR, Breitenbucher JG, Hui H, McGee D, Allen D, Martelli A, Mackman RL. Chem. Biol. 8 1107-1121 (2001)
  6. Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain. Bajaj SP, Sabharwal AK, Gorka J, Birktoft JJ. Proc. Natl. Acad. Sci. U.S.A. 89 152-156 (1992)
  7. The hydrolytic water molecule in trypsin, revealed by time-resolved Laue crystallography. Singer PT, Smalås A, Carty RP, Mangel WF, Sweet RM. Science 259 669-673 (1993)
  8. Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases. Katz BA, Luong C. J. Mol. Biol. 292 669-684 (1999)
  9. 1.59 A structure of trypsin at 120 K: comparison of low temperature and room temperature structures. Earnest T, Fauman E, Craik CS, Stroud R. Proteins 10 171-187 (1991)
  10. Synthesis and evaluation of guanidine-containing Schiff base copper(II), zinc(II), and iron(III) chelates as trypsin inhibitors. Toyota E, Sekizaki H, Itoh K, Tanizawa K. Chem. Pharm. Bull. 51 625-629 (2003)
  11. Action models for the antitumor drug camptothecin: formation of alkali-labile complex with DNA and inhibition of human DNA topoisomerase I. Streltsov SA. J. Biomol. Struct. Dyn. 20 447-454 (2002)
  12. Active site titration of bovine beta-trypsin by N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis. Sartori P, Djinovic Carugo K, Ferraccioli R, Balliano G, Milla P, Ascenzi P, Bolognesi M. FEBS Lett. 358 53-56 (1995)
  13. Automated multi-attribute method sample preparation using high-throughput buffer exchange tips. Ogata Y, Quizon PM, Nightlinger NS, Sitasuwan P, Snodgrass C, Lee LA, Meyer JD, Rogers RS. Rapid Commun Mass Spectrom 36 e9222 (2022)
  14. Crystal structure of 6-guanidinohexanoyl trypsin near the optimum pH reveals the acyl-enzyme intermediate to be deacylated. Masuda Y, Nitanai Y, Mizutani R, Noguchi S. Proteins 81 526-530 (2013)
  15. Enzyme catalysis: now you see it, now you don't. Blow D. Curr. Biol. 3 204-207 (1993)
  16. Enhancing the multi-attribute method through an automated and high-throughput sample preparation. Sitasuwan P, Powers TW, Medwid T, Huang Y, Bare B, Lee LA. MAbs 13 1978131 (2021)


Quick links