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In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors. For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa. Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1). Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa. The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain. In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa. In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised. The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases. This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design.

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, p(app), determined from the HPLC analysis of the reactions. The order of preference for the P'(1) position was estimated to be: Gly > hydrophilic, positively charged > hydrophobic, aromatic > negatively charged > Leu >>> Pro side chain. For the P'(2) position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of p(app) for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P'(3) position. The dipeptide with Pro in P'(1) did not react at all, but a tripeptide having Pro in P'(3) was a very good nucleophile. The negatively charged amino acid residues in the P'(1) position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P'(2) and P'(3) are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of alpha-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed.

Chemical modifications are a simple method to identify and modify functional determinants of enzymes. In the case of serine proteases, it is possible to induce characteristics which are advantageous for peptide synthesis. In this work, we investigated the influence of guanylation and succinylation of lysine residues on the S′-subsite specificity, the catalytic behavior and stability of trypsin. We have found, that succinylation leads to an about 10-fold better acceptance of basic residues in P1′, whereas guanylation shows no remarkable effects. Furthermore, guanylation enhances, succinylation reduces the general enzyme–substrate interactions in P2′. The structural fundamentals of these specificity changes are discussed. The catalytic behavior of trypsin was not influenced by guanylation and succinylation but an enhancement of the stability against autolytic processes by introducing additional negative charges into the protein was observed.

A peptide library consisting of di- and tripeptide esters and tripeptide amides, respectively, containing α-Tfm amino acids in different positions was synthesized and tested for enzymatic fragment condensations catalyzed by the proteases α-chymotrypsin, trypsin and clostripain.

The S1'-S3' subsite specificity of prolyl endopeptidase from Flavobacterium meningoseptum was studied by acyl transfer to libraries of amino acid amides and peptides. Whereas the S1' and S3' subsites influence the specificity for the amino component by approximately one order of magnitude, the S2' subsite possesses a markedly higher specificity. Besides the high specificity for hydrophobic residues at P1'-P3', proline was efficiently bound by the S2' and S3' subsites of the enzyme. In contrast, no binding of P1' proline-containing peptides was observed. It could be demonstrated that the specificity of the S' subsite is not restricted to L-amino acids. Effective P'-S' interactions were also found for beta- and gamma-amino acids indicating that the enzyme does not form close contacts to the backbone of P1' and P2' amino acid residues.

The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with Glu and Asp to introduce a high specificity for basic residues into the S1' site of trypsin. Both mutations cause a dramatic increase in the S1' specificity for Arg and Lys as measured by acyl transfer reactions. In K60E, the preference for Arg increases 70-fold while the preference for P1'-Lys increases 12-fold. In contrast, the preferences for other P1' residues either decrease slightly or remain the same. Thus, K60E prefers P1'-Arg over most other P1' residues by 2 orders of magnitude. Similar results are obtained when P1' specificity is measured in peptide cleavage assays. K60D exhibits an S1' specificity profile very similar to that of K60E, although the P1'-Arg preference is reduced by a factor of 2.5. Molecular modeling studies suggest that the high S1' specificity for Arg in K60E may be due to the formation of a salt bridge between Glu60 and the P1'-Arg of the substrate.

Synthesis of guanylyl(3′→5′)cytidine catalysed by RNase T1 variants (Tyr42Trp, Tyr24Trp and GluSSAla) was studied in frozen aqueous systems at-10°C and in solution at 0°C. Freezing the reaction mixture resulted in significantly enhanced dinucleoside monophosphate yields independently of the effect of mutation on substrate binding and catalytic mechanism. We assume that the protonation state of the catalytic residues is influenced by freezing, possibly due to conformational changes of the enzyme proteins.

The capability of endoproteinase Pro-C from Flavobacterium meningosepticum to form peptide bonds in frozen aqueous systems was investigated. In coupling of Bz-Gly-Pro-OMe with various amino acid amides, free amino acids, and dipeptides, freezing-induced yield enhancement strongly depended on the nucleophilic amino component ranging from 3–59%. From the different influence of freezing, it can be concluded that freeze concentration is not the only cause of yield enhancement in frozen aqueous systems. Endoproteinase Pro-C proved to be a suitable catalyst in Pro-Leu bond formation but its scope of capability in peptide synthesis is limited.

The kinetically controlled α-chymotrypsin- and trypsin-catalysed syntheses of peptides starting from simple acyl donor esters containing histidine at the P 1 -position (nomenclature according to Schechter and Berger [1] ) and lysine derivatives as amino components were examined on the basis of their kinetic parameters. Despite higher specificity constants (k cat /K M ) of trypsin-catalysed ester hydrolysis, α-chymotrypsin-catalysed acyl transfer to N e -unprotected lysine derivatives gave higher peptide yields as compared to trypsin-catalysed reactions, whereas in acyl transfer to N e -protected lysine derivatives the trypsin-catalysed reaction gave higher yields. α-Chymotrypsin-catalysed acyl transfer reactions in frozen systems demonstrated the yield-enhancing effect of freezing. Using specific ester leaving groups, both the amount of enzyme and the reaction time can be reduced. In frozen systems the e-amino function of H–Lys–OH acts as an acyl acceptor at pH ≥9.

Several dipeptides containing nonproteinogenic amino acids (especially l -tertiary leucine or l -neopentyglycine) were synthesized enzymatically. Thermolysin was used in solid-to-solid conversions in an equilibrium-controlled reaction while α-chymotrypsin was applied in kinetically controlled synthesis. The dipeptide Z-Npg-Npg-NH 2 could be obtained using a protease mixture from Streptomyces . The active enzyme has not yet been purified.

Trypsin and chymotrypsin differ strikingly in substrate specificities despite great similarity in their primary and tertiary structures. This work analyzes the role of two surface loops, loop 40 and loop 60, as structural determinants of the specificity of the S1'-subsite in chymotrypsin and trypsin. Chymotrypsin prefers P1' Arg/Lys residues, while trypsin prefers hydrophobic P1' residues. We replaced loop 40 and loop 60 in trypsin with their chymotrypsin counterparts. These mutations do not affect the S1 specificity and catalytic activity of trypsin. The S1' specificity was analyzed by monitoring acyl-transfer reactions to 16 amino acid amides. The exchange of loop 40 does not affect the S1' specificity. In contrast, the replacement of loop 60 causes a loss of specificity for P1'-Met/Ile/Leu. Combining both mutations reconstitutes a chymotrypsin-like S1' specificity. The specificity for Arg-NH2 increases 3-fold while the preferences for Met-NH2 and Ile-NH2 decrease 4- and 8-fold, respectively. Therefore, P1'-Arg/Met discrimination changes by factor 12 and P1'-Arg/Ile discrimination changes by factor 24. Thus, loop 40 and loop 60 act synergistically to determine S1' specificity in trypsin and chymotrypsin.

Recently we have demonstrated the advantage of solid- phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields. ©1997 European Peptide Society and John Wiley & Sons, Ltd.

When using proteases in direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymic peptide synthesis. Analysis of the equilibrium position reveals a favourable shift towards the peptide product if starting materials are largely undissolved in the reaction medium and the product precipitates. This approach enabled us to obtain high peptide yields in thermolysin-catalysed reactions in high-density aqueous media with an equimolar supply of substrates. The easy scale-up (up to mol-scale) of this approach is demonstrated by two examples. Z-His-Phe-NH2 and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84-88%.

The ability of the endopeptidase alpha-chymotrypsin (EC 3.4.21.1) to catalyse the reaction of various N alpha-unprotected di- and tripeptide ester derivatives with H-Leu-NH2, and with a series of C-terminal free di- and tripeptides at -15 degrees C in frozen aqueous solution was investigated. The enzyme is able to synthesize N- and C-terminal unprotected penta- and hexapeptides in up to 92% yield, depending on the amino component used, in a single-step segment-condensation reaction. Freezing the reaction mixture resulted in significantly increased peptide yields compared with the reaction at room temperature. The enzyme shows a modified nucleophilic specificity in frozen solution compared with room temperature. Nucleophilic amino components with positively charged amino acids in P2'-position are accepted.

In order to combine the high potential of frozen state peptide synthesis and the advantages of the application of immobilized proteases, the capability of carrier-bound α-chymotrypsin (CT, EC 3.4.21.1) to form peptide bonds in frozen aqueous reaction mixtures was investigated. The properties of the support materials strongly influenced the peptide yields. CT bound to rather hydrophobic supports catalysed peptide synthesis as effectively as the soluble enzyme. The immobilized enzyme preparations could be successfully re-used at least 15 times. Peptide synthesis catalysed by immobilized chymotrypsin was also studied in low water organic reaction mixtures at sub-zero temperatures. The advantages and limitations of the different approaches are discussed.

The incorporation of α-trifluoromethyl pyroglutamic acid into thyreotropin releasing hormone TRH, results in the complete stability of the p Glu-His bond against proteolytic degradation by pyroglutamyl aminopeptidase II. The influence of the trifluoromethyl group on structure and receptor affinity has been studied.

The serine proteases α-chymotrypsin and trypsin and the cysteine protease papain were used to catalyze the synthesis of Nα-protected di- and tripeptide ester substrates by acyl transfer of the corresponding Nα-protected amino acid esters to various amino acid- or dipeptide esters. The very simple and economical strategy benefits from performing the enzymatic reaction in frozen-aqueous systems. The products, which can further act as specific substrates for several important proteases, are obtainable in high yields and can be isolated easily. The results show an extraordinary shift in S' specificty of the proteases in frozen aqueous systems and are of general importance for protease-directed step-by-step peptide synthesis with a minimal protection/deprotection techniques.

α-Chymotrypsin (EC 3.4.21.1) was used for catalyzing the reaction of various N α -unprotected non-coded phenylalanine ester derivatives with H-Leu-NH 2 and H-Arg-NH 2 in frozen aqueous solution at −15 °C. Compared with reactions at room temperature, a significant yield increasing effect could be established. The kinetic parameters of ester hydrolysis show that most of the unusual acyl donors (compared with the coded phenylalanine methyl ester) are well accepted substrates for α-chymotrypsin.

Efficient enzyme-catalyzed syntheses may be carried out even when most of the starting materials and products are in the form of suspended solids. To some examples in the literature of such solid-to-solid conversions, we add the thermolysin-catalyzed reaction of X-Phe-OH (X = For, Ac, Z) with Leu-NH2 to give X-Phe-Leu-NH2. This can proceed to equilibrium yields of over 90% even in aqueous media. Thermodynamic analysis of such systems shows that solid-to-solid conversion itself will be equally favorable in any solvent at a given water activity. The reaction will normally show a “switchlike” behavior, continuing in one direction until at least one reactant has passed completely into solution. If the solid-to-solid synthesis is favorable, the equilibrium yield will be maximized by choice of a solvent in which the solubility of the starting materials is minimized. Some of the dependencies predicted by our equations can be seen in data from our own studies or in the literature. This includes a linear decrease in yield with the reciprocal of the initial amount of reactants suspended in a fixed volume.

The application of enzymes to perform selective transformations is the central aim of industrial biocatalysis. Since a simple handled C-N ligase is not available, the only alternative of general practical interest for enzymatic peptide bond formation are proteases due to the principle of microscopic reversibility. The stereo- and regiospecifitity of proteases, which guarantee racemization-free segment condensation and require only minimal side-chain protection, are significant for preparative purposes. Unfortunately, proteases are not perfect acyltransferases. Both unwanted proteolytic side reactions and the hydrolysis of the acyl-enzyme in the kinetic approach are the major problems in enzymatic peptide synthesis. The object of this paper was to demonstrate several new synthesis strategies including medium engineering that enables one to bypass unfavorable reaction routes. Planning and optimization of enzymatic peptide synthesis require the S′ subsite mapping of proteases and the knowledge of additional basic parameters that determine the reaction course.

α-Chymotrypsin was crosslinked to give a water-insoluble polymer by treatment with the bifunctional reagent glutaraldehyde. The specific activity of the crosslinked enzyme in aqueous media was three orders of magnitude lower than for the native chymotrypsin. In a medium containing more than 50% (v/v) of dimethylformamide the specific activities of both enzymes were comparable. In addition, the insoluble polymer was more stable in the presence of 60% (v/v) dimethylformamide compared with the native enzyme. Therefore, in this medium enzymatic peptide synthesis could be successfully accomplished with the crosslinked enzyme, but not with the same amount of native chymotrypsin.

Nucleophile specificities of polyethylene glycol-modified alpha-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amides both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P1' position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P2' were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P1'. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P1' > P3' > P2'; this is valid for both enzymes.

The cysteine protease clostripain, purified by affinity chromatography on a large scale, shows very high activity against BAEE using the titrimetric standard assay. Furthermore, titration of the active site with the irreversible inhibitor tosyl-lysyl-chloromethane resulted in a more than two times higher specific activity compared with literature data (Porter et al. (1971) J. Biol. Chem. 246, 7675-7682). Based on the molar enzyme concentration determined, the hydrolysis kinetics of the standard substrate BAEE were compared with those for the N alpha-protected dipeptide ester Mal-Tyr-Arg-OEt. It was demonstrated from the kinetic data that the highly purified clostripain is the most active enzyme preparation available up to now. In contrast to the standard substrate, Mal-Tyr-Arg-OEt shows a threefold lower specificity constant.

A novel method of Kyotorphin (H-Tyr-Arg-OH) synthesis without protecting and deprotecting procedures is described -α-chymotrypsin-catalyzed aminolysis of H-Tyr-OEt by H-Arg-OH in frozen mixtures. In this one-step process yields of the peptide product above 80% have been obtained.

Protease Catalyzed and Chemical Peptide Bond Formation with α-Trifluoromethyl Substituted α-Amino Acids Subtilisin, α-chymotrypsin and papain catalyzed hydrolyses of α-trifluoromethyl substituted N-benzyloxycarbonyl amino acid methylesters (Z-TFM-Xaa-OMe) 1 can be achieved only in the case of 3,3,3-trifluoroalanine. Enzymatic incorporation of Z-TFM amino acids 2 into N-terminal position of dipeptides also fails. In contrary, dipeptides with a TFM amino acid moiety in N-terminal position, e.g. TFM-Phg-L-Phe-OMe 5, react with H-Leu-NH2 to give the corresponding tripeptides 6 in high yield. Z protected dipeptide derivatives 8 with N-terminal TFM amino acids can be obtained via 4-trifluoromethyl-5-(4H)-oxazolones 7.

A new method for increasing product yield in kinetically controlled enzymatic peptide synthesis catalyzed by serine and cysteine proteases is proposed - freezing the reaction mixture. The use of the method in preparative synthesis has been demonstrated.

Chymotrypsin powder suspended in organic solvents in the presence of Na 2 CO 3 .10H 2 O catalyses peptide synthesis from X-Ala-Phe-OMe and Leu-NH 2 (X = Boc or Z). The reaction proceeds best in the most non-polar solvents, such as hexane, despite the fact that both reactants and products remain largely undissolved. Leu-NH 2 .HCl can be used in place of the free base.

In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a CN ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal α-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of CN bond formation.

Kinetically controlled chymotrypsin-catalyzed peptide syntheses are carried out using unprotected amino acids as amino components. The reactions are performed in frozen aqueous solutions. A gram scale dipeptide synthesis is described.

α-Chymotrypsin (EC 3.4.21.1) was used for catalyzing the reaction of various N α -unprotected non-coded phenylalanine ester derivatives with H-Leu-NH 2 and H-Arg-NH 2 in frozen aqueous solution at −15 °C. Compared with reactions at room temperature, a significant yield increasing effect could be established. The kinetic parameters of ester hydrolysis show that most of the unusual acyl donors (compared with the coded phenylalanine methyl ester) are well accepted substrates for α-chymotrypsin.

The incorporation of α-trifluoromethyl pyroglutamic acid into thyreotropin releasing hormone TRH, results in the complete stability of the p Glu-His bond against proteolytic degradation by pyroglutamyl aminopeptidase II. The influence of the trifluoromethyl group on structure and receptor affinity has been studied.

Fmoc-Phe-OMe,Fmoc-Ala-OMe andFmoc-Gly-OH were coupled with H-Leu-NH2 under catalytic action of chymotrypsin, papain and thermolysin, respectively. The influence of different reaction media and several reaction parameters, such as reactants and enzyme concentrations as well as reaction time, on the peptide bond formation was investigated.

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters α and β for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH2 from Mal-L-Phe-L-AlaOMe and L-LeuNH2. The experimental results demonstrate that this theory adequately describes the real process. © 1992 John Wiley & Sons, Inc.

The formation of the luteinizing hormone releasing hormone (LHRH) Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (1, from the segments Glp-His-Trp-OEt (2) and H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (3) is catalyzed by α-chymotrypsin. A yield of 71% was achieved when the reaction was performed at -15°C in a solution which contained 38 vol % DMSO and was saturated with KCI.