Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives

Shin Ono; Masato Koga; Yuya Arimura; Takahiro Hatakeyama; Mai Kobayashi; Jun-Ichi Sagara; Takahiko Nakai; Yoshikazu Horino; Hirofumi Kuroda; Hiroshi Oyama; Kazunari Arima

doi:10.3390/molecules28073150

Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives

Molecules. 2023 Mar 31;28(7):3150. doi: 10.3390/molecules28073150.

Authors

Affiliations

¹ Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan.
² Applied Bioengineering, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan.
³ Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Toyama, Japan.
⁴ Department of Applied Chemistry and Bioscience, Chitose Institute of Science and Technology, Chitose 066-8655, Hokkaido, Japan.
⁵ Department of General Education, National Institute of Technology, Ishikawa College, Tsubata 929-0392, Ishikawa, Japan.
⁶ Faculty of Science and Engineering, Setsunan University, Hirakata 572-8508, Osaka, Japan.
⁷ Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Kagoshima, Japan.

Abstract

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-pNA. 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.

Keywords: chymotrypsin; dansyl group; diphenyl α-aminoalkylphosphonate; site-selective chemical modification.

MeSH terms

Catalytic Domain
Chromatography, Liquid
Chymotrypsin* / chemistry
Tandem Mass Spectrometry*

Substances

Chymotrypsin
diphenyl

Grants and funding

This research received no external funding.