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

FEBS Letters

Milla, Paola¹  Sartori, Patrizia³  Ascenzi, Paolo²  Ferraccioli, Raffaella⁴  Balliano, Gianni¹  Bolognesi, Martino³  Carugo, Kristina Djinovic³ 

[1] Istituto di Chimica Farmaceutica Applicata, Universita'di Torino, Corso Raffaello 31, 10125 Torino, Italy;Dipartimento di Scienza e Tecnologia del Farmaco, Universita' di Torino, Via Pietro Giuria 9, 10125 Torino, Italy;Dipartimento di Genetica e Microbiologia, Universita' di Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy;CNR, Centro Sintesi e Stereochimica di Speciali Sistemi Organici, Via Golgi 19, 20133 Milano, Italy

关键词: Bovine β-trypsin;    Nα -(N;    N-Dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester;    Dmc-azaLys-ONp;    Enzyme kinetics;    Active enzyme concentration (determination of);    Acyl enzyme adduct (X-ray crystal structure of);    trypsin;    bovineβ-trypsin;    pN-pGB;    p-nitrophenyl p-guanidinobenzoate;    Z-Arg-ONp;    N-α-carbobenzoxy-l-arginine p-nitrophenyl ester;    Z-Lys-ONp;    N-α-carbobenzoxy-l-lysine p-nitrophenyl ester;    Dmc-azaLys-ONp;    Nα -(N;    N-dimethylcarbamoyl)-α-aza-lysine p-nitrophenyl ester;    Dmc-azaLys;    Nα -(N;    N-dimethylcarbamoyl)-α-azalysine;   

DOI  :  10.1016/0014-5793(94)01366-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Kinetics of bovine β-trypsin (trypsin) with the N ^α -(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 · N ^α -(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.

【 授权许可】

Unknown