Active site titration of bovine β-trypsin by Nα-(N,N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis

Patrizia Sartori, Kristina Djinovic Carugo, Raffaella Ferraccioli, Gianni Balliano, Paola Milla, Paolo Ascenzi, Martino Bolognesi

Research output: Contribution to journalArticlepeer-review

Abstract

Kinetics of bovine β-trypsin (trypsin) with the Nα-(N,N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0°C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin · Nα-(N-N-dimethylcarbamoyl)-α-aza-lysine acyl · enzyme adduct has been solved by X-ray crystallography at 2.0 Å resolution (R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue.

Original languageEnglish
Pages (from-to)53-56
Number of pages4
JournalFEBS Letters
Volume358
Issue number1
DOIs
Publication statusPublished - Jan 16 1995

Keywords

  • Active enzyme concentration (determination of)
  • Acyl enzyme adduct (X-ray crystal structure of)
  • Bovine β-trypsin
  • Dmc-azaLys-ONp
  • Enzyme kinetics
  • N-(N,N-Dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Genetics
  • Molecular Biology
  • Structural Biology

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