| Autor: |
Ono S; Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Koga M; Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Arimura Y; Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Hatakeyama T; Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Kobayashi M; Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Sagara JI; Applied Bioengineering, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan., Nakai T; Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Toyama, Japan., Horino Y; Department of Applied Chemistry and Bioscience, Chitose Institute of Science and Technology, Chitose 066-8655, Hokkaido, Japan., Kuroda H; Department of General Education, National Institute of Technology, Ishikawa College, Tsubata 929-0392, Ishikawa, Japan., Oyama H; Faculty of Science and Engineering, Setsunan University, Hirakata 572-8508, Osaka, Japan., Arima K; Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Kagoshima, Japan. |
| Abstrakt: |
We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N -hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe P (OPh) 2 ]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-( R )-Phe P (OPh) 2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe- p NA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman-Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin. |
