Allosteric site of serine proteinases: localization, functional role and manifestations in vitro

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SI "O.S. Kolomiychenko Institute of Otolarynglology, NAMSU"

ALLOSTERIC SITE OF SERINE PROTEINASES: LOCALIZATION, FUNCTIONAL ROLE AND MANIFESTATIONS IN VITRO

Verevka Serhij Viktorovych

Doctor of Biological Sciences, Professor,

Head of the Department of Biochemistry

Kyiv

Summary

serine proteinase molecular allosteric

The study of molecular mechanisms of regulation of biologically active molecules is a necessary condition for understanding the course of the processes mediated by them. Serine proteinases are a large group of enzymes that play a leading role in the regulation of numerous physiological and pathogenic processes. Like many enzymes, SP are allosteric ones. In addition to the active center, they contain the region, whose interaction with corresponding compound changes the activity of enzyme. Various compounds can act as allosteric effectors. They may be substrates, substrate-like compounds, and mimics of some fragments of substrates. Taking into account the importance of serine proteases'functions, it's deserve attention the systematic data on localization, functional role and manifestations of allosteric region.

Key words: proteolysis, serine proteinases, allosteric site.

The main text

At common structure of the catalytic centers, the action of serine proteinases SPs differs in the direction of the splitting. This difference determines the performance of each individual enzyme of its physiological functions. The simplest in them both by structure and catalytic properties are SPs of pancreas - chymotrypsin, trypsin and elastase (E.C. 3.4.21.1, 3.4.21.4 and 3.4.21.11, respectively). They are characterized by close similarity of molecular mass, high homologies of sequences and tertiary structures of molecules. The kinetic parameters of these enzymes are quite similar. However, they differ in substrate specificity. Trypsin hydrolyzes specifically bonds formed by the carboxyl group of lysine or arginine; chymotrypsin breaks down phenylalanine, tyrosine and tryptophan, elastase is characterized by hydrolysis of small amino acids of hydrophobic nature - such as alanine and, to a lesser extent, valine. This difference is due to small differences in the structure of the "hydrophobic pocket" that binds the side chain of the amino acid, whose carboxyl group forms a cleavaged peptide bond [1 -3]. The corresponding residue and binding site are denoted as P1 and S1, by conventional Schechter-Berger nomination (Fig. 1).

Fig. 1 Placement of the polypeptide chain at the interaction with enzyme according to Schechter-Berger nomination [4]. The arrow indicates the peptide bond that is being cleaved

Like many enzymes, SPs are allosteric ones. A large number of studies have been devoted to the structure and role of their catalytic centers and binding sites. However, no less interesting is the allosteric one, which is involved in the regulation of various SPs-mediated processes both in vivo and in vitro. It should be emphasized that the data on the allosteric site are extremely discrete. Historically, the first of the detected manifestations of the allosteric region of SPs was substrate activation, which is a nonlinear increase in enzyme activity with increasing of substrate concentration. At the study of trypsin hydrolysis of low-molecular-mass substrates, the lack of correlation between the structures of the substrate and the effector was shown [5]. Thus, at low concentrations N-carbobenzoxy-L-arginyl ethyl ether is a better substrate than N-tosyl-L-arginyl ethyl ether, and at higher - due to the activating action of the substrate - vice versa. Substrate activation was observed at the hydrolysis by trypsin, thrombin and plasmin methyl esters of N-tosyl-L-lysine and N-tosyl-L-arginine, and for each of these enzymes it was detected differently [6]. Substrate activation was also observed during leukocyte elastase hydrolysis of a group of substrates of the N-succinyl-(L-alanine)n-para-nitroanilide type, where n ranged from 2 to 5 [7]. At the hydrolysis of LMM substrates with a-chymotrypsin substrate activation is much less pronounced. The non-competitive nature of hydrolysis by a-chymotrypsin of N-acetyl-L-tyrosine-ethyl ether in the presence of Nbenzoylor N-tosyl-L-arginine-methyl esters was considered as an exception to this rule. Subsequently, however, such exceptions became more and more, which eventually led to the conclusion that the substrate-effector modification of MichaelisMenten kinetics is widespread and that this phenomenon is characteristic of a - chymotrypsin, too [8].

In addition to substrates, a large number of compounds are capable for allosteric activation of SP. Thus, tryptic hydrolysis of short-chain esters of acyl-amino acids is accelerated in the presence of alkylammonium ions [9-11]. N-dansyl-Larginine also exhibits the properties of allosteric trypsin effector due to the binding both to the active site of the enzyme and outside it [12]. Salts of trialkylammonium derivatives of azobenzene accelerate a-chymotrypsin-mediated hydrolysis of Nacetyl-amino acid anilides [13]. Hydrolysis by a-chymotrypsin of a nonspecific substrate - acetyl-p-nitrophenyl ether - is accelerated by a large number of hydrophobic compounds [14,15]. The activation region of chymotrypsin is thought to be similar to the binding site, although more polar, and located somewhere on the surface of the enzyme molecule [13]. Data on reactions in such kind of systems suggest that a protein capable of simultaneously interacting with both enzyme's binding site and allosteric one will be subject to much faster hydrolysis than in the case of a single interaction [8]. The latter assumption was a kind of key for determining of the location and functional role of SPs' allosteric site.

It is known that the limitation of the hydrolytic action of proteinases is mediated by protein inhibitors. These compounds are sharply superior to conventional protein substrates relatively to the affinity to enzyme's active center. Therefore, they are sometimes called as even the ideal substrates for the corresponding enzymes [16]. This leads to increased interest in the area of the direct contact between the enzyme and the inhibitor. Protein inhibitors of serine proteinases are grouped into at least two dozen families. Inhibitors of one family have an extremely high degree of structural homology, while differences between families are quite significant [17,18]. At the same time, the conformations of the reactive centers of inhibitors (P3...-...P3') of different families are very similar [365,410]. Like substrates, the specificity of protein inhibitors is determined, even not so strictly, by the nature of the amino acid residue located at the P1 position of the reactive center. Replacement of the amino acid in this position leads to a corresponding change in the specificity of the inhibitor [19-21]. Most protein inhibitors are characterized by a rigidly fixed structure by disulfide bonds. The main purpose of this fixation is to maintain the reactive center's conformation in the optimal state for the interaction with enzyme. Any factors, that disrupt the conformation of the reactive center, lead to a sharp decline in the inhibitory properties [21 -25]. The richest material for consideration is given by the results of X-ray structural studies of SPs and protein inhibitors both individually and

Fig. 2 Spatial model of the complex of soybean trypsin inhibitor with trypsin [26]

The formation of the enzyme-inhibitory complex is accompanied by masking both in the enzyme and in the inhibitor of almost identical surfaces with a total area of about 1500 A². In this area the share of six residues P3 -...P3' reactive center accounts for 70 to 77% of the surface inhibitor that is masked by complexation [27,28]. In this case, amino acid residues at P1and P2'positions have an increased degree of masking and an abnormally high number of contacts with amino acid residues of the enzyme [27,28]. The results of X-ray diffraction analysis of the complex of soybean trypsin inhibitor with trypsin suggested that the amino acid residue at the P2'-position plays no less value than the residue at the P1 -position in the formation of the enzyme-inhibitory complex [26]. At the same time for P2'positions of different families of protein inhibitors a pronounced dominance of hydrophobic amino acids was found by numerous authors [29-31]. Neither for P1'nor for P3'-positions any such regularities are observed. At the study of the kinetic parameters of a number of highly homologous ovomucoids, it was noted that the replacement in the P2'-position of tyrosine by less hydrophobic histidine leads to a decrease in inhibitory properties, while the ovomucoid with aspartic acid in this position has no protein inhibitor properties at all [16]. The comparison of inhibitory properties of the first domain of the SBI-D-II inhibitor and the second domain of the SBI-C-II inhibitor shows the same pattern, since the reduction of the hydrophobicity at the P2'-replacer (Met ^ Gln) sharply reduces the inhibitory properties [32]. A similar effect was observed at the oxidation of methionine at the P2'-position of the SBI-C-II inhibitor by hydrogen peroxide [33]. A large number of such data led to an attempt to experimentally test the role of the S2'-site in the processes of intermodular SPs' interactions.

To test this assumption, the enzyme-fixed probe method was used, in which the capture of one hydrophobic nucleus by the "hydrophobic pocket" of the enzyme created the preconditions for placing the second one into the study area (Fig. 3)

Fig. 3 Reactive center of Bowman-Birk chymotrypsin inhibitor and the structure of 1,5-bis-dibenzylamino-pentane [34]

With this arrangement, the demands for the binding parameters of S₂'-site may be incomparably lower than for "hydrophobic pocket" S1. It is easy to calculate that the binding of one of the hydrophobic nuclei by S1 -site creates a local ligand concentration of the order of 300 mM in a volume with a radius equal to the length of the free part of the examined molecule [35]. The revealed effect of such compounds on the hydrolytic action of chymotrypsin on various substrates allowed to unambiguously identify the S2'-site as a long-known, but unidentified, allosteric one. Moreover, for the first time an explanation of its functional role was given. Synchronous interaction of the binding Siand allosteric S^-sites with properly conformed and adequate by ligand specificity Piand P2-residues ensures the effective binding of the enzyme to the reactive center of the protein inhibitor [36]. Rigid fixation of residues P1 and P2' in the optimal (canonical) conformation provides effective complexation not only with the corresponding proteinases, but also with their anhydro-derivatives, to which the serine of the hydrolytic center is converted into dehydroalanine. The stability of such complexes is comparable to the complexes formed by active enzymes [37,38]. The exceptions to this rule are plasma inhibitors a,1-antitrypsin and a,2-antiplasmin. They are belonging to the serpine family, whose complexes with anhydro-enzymes are unstable [39,40], while they bind covalently to active enzymes [41]. Unlike most protein inhibitors, the reactive centers of serpines are mobile, passing through a "canonical conformation" [42]. Therefore, serpines differ not only in the dynamics of complex formation, but also in that it is mediated by the formation of a covalent adduct with a split reactive center. At the absence of such an interaction no strong complexation should occur, as is observed at the case of interaction of serpines with anhydro-derived enzymes [39,40].

Considered mechanism is also involved in the interaction of serine proteinases with a2-macroglobulin. It is the single protein inhibitor that binds proteinases outside the hydrolytic center. The enzyme retains the ability to interact with low molecular weight substrates and inhibitors. The capture of proteinase by the macromolecule a₂--macroglobulin is due to conformational changes caused by cleavage in the loop of the so-called bait site in a small section of the sequence -Arg*-Leu-Val*-His-Val-. Trypsin, plasmin and thrombin cleave the bond formed by arginine, while elastase cleaves the valine bond [43,44]. In both cases, the P2'-positions of the corresponding cleavages a replaced by hydrophobic amino acid.

Thus, the allosteric region of serine proteinases plays a key role in the limitation of proteolytic processes. It provides increased affinity of the active center of the enzyme to the reactive centers of protein inhibitors. However, this type of interaction does not exhaust the functional roles of the allosteric site. It is known that trypsinlike proteinases provide activation of various protein proforma due to highly selective cleavage of the so-called activation cleavage sites. Examination of the primary sequences of such sites indicates a pronounced dominance in their P2'positions of hydrophobic amino acids, less often - positively charged [45]. Rapid and selective cleavage of activation linkages in precursor proteins correlates well with the assumption of accelerated hydrolysis in the case of protein interaction with both the main and effector regions of the enzyme [8]. No less significant is the method for determination of trypsin by hydrolysis ща protamine [46]. The high content of arginine in this peptide provides statistically the filling of both the binding site and the allosteric one by adequate ligands. The high efficiency of hydrolysis of such substrate provides the determination of minimal amounts of trypsin-like proteinases.

Very indicative in this respect are the areas of activation cleavage of proteinaseactivated receptors (PARs) (Fig. 4).

Fig. 4 Sequences of activation cleavage sites of PARs' The arrows indicate the bond to be broken [47]

PARs are activated due to proteolytic cleavage by thrombin and other trypsinlike SPs of the N-terminal preactivation peptide, which converts the remaining part of the extracellular fragment into a ligand that covalently bound to the receptor molecule. Effective functioning of such a system is possible only at exceptionally high affinity of the activator to the cleavage site of receptor. In other words, the structure of this site must meet the same structural and functional requirements as for the sites of cleavage of protein proforma. That's why the allosteric site is involved in the regulation of processes of a more complex level than the restriction of proteolysis and activation of proteins' proforms.

Along with participation in the above regulatory processes, the allosteric site has a long list of manifestations in vitro. It is known that the enzymatic cleavage of the peptide chain goes through the stage of formation of the acyl-enzyme. The amino component of the cleavable chain is released. That is, the remaining group and the leaving one are formed. The study of the substrate specificity of proteases was conducted mainly in the side of remaining group, while the role of interactions with leaving one was ignored. Finding out the location and role of the allosteric site explains a number of anomalies observed in the study of the interaction of SPs with numerous low-molecular-mass compounds. In particular, the dynamics of thrombin's interaction of with similar compounds is determined dramatically by the hydrophobicity of the complementary for S2'-site substituent [48]. N-benzoyl-Ltyrosine-amide is not hydrolyzed by a-chymotrypsin. This can be explained by the advantage of unproductive binding with the orientation of the hydrophobic substituent towards the allosteric site. N-benzoyl-L-tyrosyl-glycine-amide, which has a more bulky leaving group, is cleaved quite efficiently. At the additional presence of the D-isomer in the system, hydrolysis is blocked due to the ability of the latter to block the active site by unproductive binding. A common substrate for the study of trypsin-like activity - N-benzoyl-L-arginine para-nitroanilide - in the presence of the D-isomer undergoes much more efficient cleavage. This can be explained by the ability of the D-isomer to bind to the allosteric region without creating steric barriers to interaction of the active site with L-isomer. On this reason the D-isomer is an allosteric activator of hydrolysis of the L-one [49,50].

The participation of the allosteric site in the interaction of serine proteinases with affinity sorbents is not without interest, too. Affinity chromatography is based on the ability of biological macromolecules to bind specifically immobilized ligands, that are complementary to their binding sites. In the case of the simplest SPs the affinity-binding site is the "hydrophobic pocket" S1, which determines the substrate specificity of the enzyme. The optimal for affinity binding are the interactions with dissociation constant of the order of 10^-5-10^-6 M [51]. Theoretically, interactions with Kd greater than 10^-3 M are not important for affinity sorption-desorption processes, because instead of sorption only inhibition should occur - protein yield in the form of a smeared peak commonly and immediately after inactive material [52]. However, for the case of SPs a number of violations of this rule are known. Thus, the classic ligand for affinity isolation of chymotrypsin-like enzymes is the methyl ester of Dtryptophan (Kd interaction of a-chymotrypsin with D-tryptophan derivatives in solution exceeds 2 mm [53]). Immobilization of this ligand on the activated matrix directly does not give satisfactory results, while the use of a spacer produces a sorbent that provides 100% yield of purified chymotrypsin [51]. No less traditional ligand is 1,4-phenyl-butyl-amine. Its interaction with a-chymotrypsin in solution is characterized by a Kd 2.8 mm [54]. Affinity sorbents with his ligand bind effectively chymotrypsin only after the concentration of the immobilized ligand reaches a certain threshold level. Affinity sorbents based on L-phenylalanine (Kd with chymotrypsun un solution is 2 mM) show similar properties [55]. In all three cases desorption is achieved only with the use of a "deforming eluent". A similar pattern is observed in the case of immobilized para-amino-benzamidine. When immobilizing this ligand directly on the insoluble matrix there is only inhibition of trypsin passage through the column is observed. This consists fully with the theory of affinity chromatography. The use of a spacer leads to the formation of a sorbent from which trypsin can be desorbed by "deforming eluent" only. That allow us to conclude, that the role of the spacer is not limited to the generally accepted increase in steric availability of immobilized ligand [51]. According to the above data, the sharp increase in the binding properties of sorbents is a consequence of the transition to the binding of the enzyme to the sorbent by bi-ligand mechanism with participation of allpsteric site. A similar effect results in enhanced of chymotrypsin binding by immobilized phenyl-butyl-amine in the presence of soluble low molecular weight hydrophobic compounds [56]. It is worth mentioning the changes in the binding properties of a number of sorbents depending on the structure of immobilized dinuclear ligands, too [57]. It should be noted that the promoting effect of allosteric region is inherent in all three stages of enzyme interaction with the substrate - the formation of non-covalent Michaelis complex, the transformation of the latter into acyl enzyme and hydrolysis of the latter [58]. It is worth mentioning the promotion by low-molecular mass hydrophobic compounds of hydrolysis of chymotrypsinformed acyl enzymes [37]. The possibility of alternative interactions of enzymes with substrates and products can interfere noticeably on the flow of the reaction [59].

Conclusion

Submitted data prove for the important role of allosteruc sute in the regulation of SPs-mediated processes. The aim of this work was to present the most striking manifestations of the action of this site, which corresponds to the S2'site by Schechter-Berger nomination. There is no doubt that the list of manifestations of the allosteric site of serine proteases, both in vivo and in vitro, will be constantly updated. There is no doubt that taking into account the role of the allosteric site is a necessary condition for creating effective means of influencing proteinase-mediated processes.

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