Malaysian Journal of Analytical Sciences Vol 24 No 5 (2020): 630 - 635

 

 

 

 

SYNTHESIS OF SEMISYNTHETIC TRYPSIN-1,10-PHENANTHROLINE COMPLEXES WITH DIVALENT METAL IONS FOR HYDROLYSIS OF AZOCASEIN

 

(Sintesis Kompleks Separa Sintetik Tripsin-1,10-Fenantrolin dengan Ion Logam Divalen untuk Hidrolisis Azokasin)

 

Mohd Basyaruddin Abdul Rahman^1,2*, Azizah Misran², Muhammad Alif Mohammad Latif^1,2, Emilia Abdulmalek^1,2

 

¹Integrated Chemical BioPhysics Research

²Department of Chemistry, Faculty of Science

Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

 

*Corresponding author: basya@upm.edu.my

 

 

Received: 30 March 2020; Accepted: 2 September 2020; Published: 12 October 2020

 

 

Abstract

Modification of trypsin from bovine pancreas was studied to understand the biomolecular interactions between the protein and ligand, toward metal ion. A semisynthetic complex of trypsin-1,10-phenanthroline (trypsin-PHN) was prepared and investigated for its role in the hydrolysis of azocasein. Predicted results from molecular docking studies aid in the comprehension of the protein-ligand system. PHN ligand demonstrated the ability to provide more sites for interactions with metal ions and contribute extensively to the development of a new generation of industrial biocatalysts. The trypsin-PHN complex had an increment of 40% activity in the hydrolysis of azocasein. In the presence of 5 mM Ca²⁺ ions the activity was higher than native enzyme but decreased in the presence of Mg²⁺, Zn²⁺ and Fe²⁺ ions, thus, providing additional insight into potential inhibitors of the rational enzyme design.

 

Keywords:  metalloenzyme, semisynthetic, trypsin, biocatalyst, hydrolysis

 

Abstrak

Modifikasi tripsin daripada pankreas anak lembu telah dikaji untuk mengetahui interaksi biomolekul di antara protein dan ligan, dan ion logam. Satu kompleks separa sintetik tripsin-1,10-fenantrolin (trypsin-PHN) telah disediakan dan aktiviti terhadap hidrolisis azokasin telah dikaji. Hasil jangkaan daripada kajian pendokkan molekul turut membantu dalam kajian sistem protein-ligan ini. Ligan PHN menunjukkan kebolehan untuk memberikan lebih tapak interaksi dengan ion logam yang berkeupayaan mencetus pembangunan dalam generasi baru industi biomangkin. Kompleks tripsin-PHN menunjukkan peningkatan aktiviti sebanyak 40% dalam hidrolisis azokasin. Kehadiran sebanyak 5 mM ion Ca²⁺ turut meningkatkan aktiviti berbanding enzim asal tetapi menurun dengan kehadiran ion Mg²⁺, Zn²⁺ dan Fe²⁺. Maklumat ini dapat membantu penghasilan perencat dalam rekabentuk enzim secara rasional.

 

Kata kunci:  metaloenzim, separa sintetik, tripsin, biomangkin, hidrolisis

 

References

1.      Qi, D., Tann, C. M, Haring, D. and Distefano, M. D. (2001). Generation of new enzymes via covalent modification of existing proteins. Chemical Reviews, 101: 3081-3111.

2.      Mayer, C., Gillingham, D. G., Ward, T. R. and Hilvert, D. (2011). An artificial metalloenzyme for olefin metathesis, Chemical Communications, 47: 12068-12070.

3.      Heinisch, T. and Ward, T. R. (2010). Design strategies for the creation of artificial metalloenzymes, Current Opinion in Chemical Biology, 14(2), 184-199.

4.      Lu, Y., Yeung, N., Sieracki, N. and Marshall, N. M. (2009). Design of functional metalloproteins. Nature, 460, 855-862.

5.      Abdul Rahman, M. B. and Latif, M. A. M. (2019). Interaction studies of putative chemical ligands in binding sites of thermostable lipase from Geobacillus zalihae strain T1. Malaysian Journal of Analytical Sciences, 23(4): 613-624.

6.      Abdul Rahman, M. B., Jaafar A. H, Basri, M., Raja Abdul Rahman, R. N. Z. and Salleh, A. B. (2014). Biomolecular design and receptor-ligand interaction of a potential industrial biocatalyst: A thermostable thermolysin-phosphoeth-anolamine-Ca²⁺ protein complex. Journal of Advanced Catalysis Science and Technology, 1: 1-5.

7.      Villalonga, R., Villalonga M. R. and Gomez L. (2000). Preparation and functional properties of trypsin modified by carboxymethylcellulase. Journal of Molecular Catalysis B: Enzymatic, 10: 483-490.

8.      Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.

9.      Tomarelli, R. M., Charney, J. and Harding, M. L. (1949). The use of azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity. Journal of Laboratory and Clinical Medicine, 34: 428-433.

10.   Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J. (1998). Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function, Journal of Computational Chemistry, 19: 1639-1662.

11.   Walczak, M. M. and Flynn, N. T. (1998) Spectroelectrochemical study of the generation of tris-(1,10-phenanthroline)iron(II/III) from μ-oxo-bis[aquabis(1,10-phenanthroline)iron(III)]. Journal of Electro-analytical Chemistry, 441(1–2): 43-49.

12.   Bao, T. and Pawlyszyn, J. (2006). Role of calcium binding in protein structural changes and phospholipid–protein interactions studied by capillary isoelectric focusing with whole column imaging detection, Analytica Chimica Acta, 559(1): 1-8.

13.   Abdul Rahman, M. B., Misran, A., Basri, M., Raja Abdul Rahman, R. N. Z., Salleh, A. B. and Abdul Wahab, H. (2005). Screening and docking chemical ligands onto pocket cavities of a protease for designing a biocatalyst. Biocatalysis and Biotransformation, 23: 211-216.

14.   Ananthanarayanan, V. S. and Kerman, A. (2006). Role of metal ions in ligand-receptor interaction: insights from structural studies. Molecular and Cellular Endocrinology, 246: 53-59.

15.   Katz, A. K., Glusker, J. P., Beebe, S. A. and Bock C. W. (1996). Calcium ion coordination:  A comparison with that of beryllium, magnesium, and zinc. Journal of American Chemical Society, 118(24): 5752-5763.

16.   Steinreiber, J. and Ward, T. R. (2008). Artificial metalloenzymes as selective catalysts in aqueous media. Coordination Chemistry Reviews, 252: 751-766.