Allosteric modulation of primary specificity of serine proteinases

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Allosteric modulation of primary specificity of serine proteinases

Malezhyk Anastasia Olegovna

Student

O.O. Bogomolets National Medical University, Ukraine

Voroshylova Natalia Mikhailivna

Candidate of Biological Sciences, Senior Lecturer, Major Researcher O.O. Bogomolets National Medical University, Ukraine, SI «O.S.Kolomiychenko Institute of Otolarynglology, NAMSU», Kyiv, Ukraine

Obernikhina Nataliya Velcdymyrivna

Candidate of Chemical Sciences, Associate Professor O.O. Bogomolets National Medical University, Ukraine

Summary

The selectivity of serine proteinases action is mediated by high-specifice binding of the proper parts of the protein substrate. Among such protein targets a special place belongs to the areas of functionally conditioned interaction with the active center of the enzyme. Their sharp difference in enzyme affinity is due to synchronous interaction of the binding and allosteric sites of the active site with specific amino acid residues of the substrate that are adequate in specificity and placed in the proper conformation. This paper is devoted to the consideration of the manifestation of allosteric erosion of primary specificity of serine proteinases. Examples of such effects as well as the possibilities of their application are discussed.

Keywords: proteolysis, serine proteinases, specificity, allosteric site.

Serine proteinases form a large family of enzymes involved in countless physiological and pathophysiological processes. Each of the enzymes performs their functional role due to substrate specificity, i.e. the ability to recognize and interact with a specific bond of the target protein. The simplest serine proteinases are pancreatic enzymes - trypsin, chymotrypsin and elastase (E.C.3.4.21.1,3.4.21.4 and 3.4.21.11, respectively). They are characterized by close values of molecular weights, high degrees of homology of primary sequences and tertiary structures. The kinetic parameters of these enzymes are quite similar, too. The basis for the difference between them is their primary substrate specificity. Trypsin splits specifically the bonds formed by the carboxyl group of positively charged amino acids, chymotrypsin splits on large hydrophobic amino acids, elastase is characterized by hydrolysis of small hydrophobic ones. This is due to differences in the structure of the "hydrophobic pocket" that connects the side chain of the corresponding amino acids [1]. The chymotrypsin molecule has a recess with dimensions of 12x6.5x4A, which is able to bind large hydrophobic radicals. This allows you to place in it the aromatic residue of Phe, Tyr, Trp, Leu or Ile. In the trypsin molecule at the bottom of a similar pocket instead of Serisg is Aspisg. Its carboxyl group forms an ionic bond with the positively charged amino- or guanidine groups of Lys or Arg. In the case of elastase, the two glycines at positions 216 and 226 are replaced by Vabi6 and Thr226. This ensures that only small Ala or Val side chains are connected. That's why the primary specificity of the simplest serine proteinases is determined by the parameters of the "hydrophobic pocket". According to the generally accepted Schechter &-Berger nomenclature, it is designated as S1 (Fig. 1).

Similarly to numerous enzymes, serine proteinases are allosteric ones. Their allosteric site corresponds to S2' subsite according to Schechter & Berger nomenclature. The interaction of this site with complementary ligand affects the catalytic properties of the enzyme and leads to an increase in its binding and catalytic properties. Synchronous interaction of the binding and allosteric regions of the enzyme with amino acid residues in P1 and P2' positions of the target bond the highest mutual affinity between enzyme and substrate.

Fig. 1. The placement of polypeptide chain at the interaction with enzyme by Schechter &-Berger [2]. The arrow indicates the peptide bond that is being cleaved.

allosteric modulation serine proteinases

The mutual placement of these residues can either be fixed in the optimal canonical conformation, or be in a state of dynamic equilibrium with it [4]. Thus, in most cases of interaction of serine proteinases with reactive centers of protein inhibitors and sites of activative cleavage of pro-forms of various proteins, the P1 position is occupied by an amino acid residue corresponding to the primary specificity of the enzyme. Hydrophobic or positively charged residues are placed in their P2'-positions [5].;

However, such interaction leads to some kind of erosion of the primary specificity of serine proteinases. Thus, the main pancreatic trypsin inhibitor (Lys*- Ala-Arg reactive center) effectively blocks not trypsin only but chymotrypsin, too [6]. In contrast, the a1-proteinase inhibitor (Met*-Ser-Ile reactive center) effectively blocks elastase, trypsin, and chymotrypsin [7]. Affinity sorbents with spatially dispersed hydrophobic ligands (Fig. 2) effectively bind not chymotrypsin-like enzymes only but also trypsin-like ones.

These and similar examples show that with the full participation of the allosteric site in the interaction of serine proteases with complementary structures the substituent in the P1 position does not play a crucial role. This significantly expands the spectrum of the action of corresponding proteinases, providing blockade of the enzyme or its activation cleavage in unexpected areas.

Fig.2. Affinity ligands with spaced hydrophobic groups [8].

References:

[1] Fersht, A. Enzyme Structure and Mechanism. 2nd Edition (1984). W.H.Freeman, New York, 475 p.

[2] Schechter, I., Berger, A. (1967). On the size of the active site in proteinases. I. Papain. Biochem. Biophys. Res. Communs., 27(2), 157-162.

[3] Dobo, J., Gettins, P. (2004). a1-proteinase inhibitor forms initial non-covalent and final covalent complexes with elastase analogously to other serpin-proteinase pairs. J.Biol.Chem. 279(10), 9264-9269.

[4] Verevka, S.V. (20220. Allosteric site of serine proteinases: location, functional role and manifestations in vitro. Grail of Science. 12-13, 188-197. DOI 10.36074/grail-of- science.29.04.2022.029

[5] Laskowski, M., Empie, M., Kato, I., et al. (1981). Correlation of amino acid sequence witj inhibitor activity and sprcificity of protein inhibitors of serine proteinases. Coll.Les.Biol.Chem. 9(32), 136-152.

[6] Blow, D., Wright, C. (1997). A model for the association of bovine pancreatic trypsin inhibitor with chymotrypsin and trypsin. J. Mol. Biol. 69(1), 137-44.

[7] Kaslik, G., Kardos, J., Szabo, E., et al. (1997). Effects of serpin binding of the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site. Biochem. 36(18), 5455-5464,

[8] Kolodzeiskaya, M., Verevka, S. (1990). Comparative study of the inherent properties of serine proteases of lower and higher vertebrates. Ukr. Biochem. Journ. 62(6), 31 -37 (in Russian).

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