RESUMEN

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The reverse action of a trypsin-free elastase isolated from porcine pancreas was studied in frozen aqueous systems. Under frozen state conditions, porcine pancreatic elastase was able to catalyse peptide bond formation more effectively than in solution at room temperature. The acceptance of free amino acids as nucleophilic amino components indicates a changed specificity of the endoprotease in frozen reaction mixtures. In elastase-catalysed formation of Ser-, Ile- and Val-X-bonds in frozen aqueous reaction mixtures, peptide yields obtained depended on the P1 amino acid and the acyl donor chain length.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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%.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

A series of model peptides containing alpha-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease alpha-chymotrypsin was synthesized by solution methods to investigate the influence of alpha-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted to the P1 position and still considerable proteolytic stability for peptides substituted at the P2 and P'2 positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid alpha-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the alpha-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P'1 substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the alpha-Tfm group can be outweighed by an advantageous interaction of the flourine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer.

Asunto(s)

RESUMEN

Freezing of the reaction mixture is a powerful tool in proteinase-catalysed peptide synthesis. In this study, the considerable yield-increasing effect of freezing has been analysed by physical and analytical methods. 1H-NMR relaxation time measurements have been used to determine the amount of unfrozen water in partially frozen systems thus quantifying the extent of the 'freeze concentration effect' for the first time. Comparative studies in ice and at room temperature verify the importance of freeze-concentration which, however, is not sufficient for a complete understanding of the observed effects. Furthermore, the phase behaviour of frozen systems is discussed.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

We have studied ribonuclease T1 (EC 3.1-27.3)-catalysed synthesis of guanylyl-(3'-->5')cytidine in frozen aqueous reaction mixtures at -10 degrees C and in solution at 0 degree C in order to investigate whether ribonuclease-catalysed synthetic reactions can take advantage of the yield-increasing effect of freezing as was reported for protease-catalysed peptide synthesis. Under frozen state conditions, substantially increased yields of GpC were obtained compared to the reactions in solution. From the fact that no irreversible hydrolysis of the 2'3'-cyclic donor was observed it can be concluded that transesterification of the newly formed phosphodiester bond is the most important yield-limiting factor in ribonuclease T1-catalysed dinucleoside phosphate synthesis.

Asunto(s)

RESUMEN

The importance of electrostatic interactions between charged residues at the P3 position of substrates and the S3 subsite of the cysteine protease clostripain was investigated. For this purpose quantitative enzymatic hydrolysis studies using steady state kinetics have been carried out within a set of N alpha-protected synthetic dipeptide ester substrates with systematic changes of their charge in the P3 position. It was demonstrated that, in contrast to the former postulated second anionic S3 subsite, the lowest specificity was for the hydrolysis of the positively charged substrates. However, this effect was strongly dependent on the individual amino acid at P1. Furthermore, we investigated how far these P3-S3 interactions reflect on the S' subsite specificity via acyl transfers. Apart from the general weak influence of the charge at P3 on the deacylation kinetics, nucleophiles with proline at P'1 play an extraordinary role. Surprisingly, in contrast to the poor primary lysine specificity, acyl transfer using P1 lysine substrates does not affect the nucleophile efficiency found with the corresponding arginine substrates.

Asunto(s)

RESUMEN

During the past decade proteases have been widely used as catalysts in peptide synthesis. Unfortunately, they are not ideal ligases. Enzymatic peptide synthesis in frozen aqueous systems has been developed as an approach towards the suppression of competitive reactions. This paper summarizes reports concerning the behaviour of non-enzymatic as well as of enzyme-catalysed reactions when the reaction mixture is frozen. The advantages of freezing the reaction mixture in serine and cysteine protease-catalysed peptide synthesis, the influence of modified reaction conditions and the possible reasons for the yield-increasing effect of freezing are discussed.

Asunto(s)

RESUMEN

One of the attractions of using enzymes for chemical syntheses is the control of stereochemistry: problems of racemization that attend chemical C-N ligation methods are completely avoided. Furthermore, the enzymatic approach has the advantage that only minimal protection-deprotection steps are involved. The Impetus to develop non-conventional catalysis procedures has sprung from the lack of usable native enzymes that normally catalyze the formation of peptide bonds for biotransformation. In peptide syntheses that make use of the 'reverse hydrolysis potential' of proteases several problems need to be considered, especially the necessity of minimizing competing hydrolysis of weakly activated acyl donor esters and the need to circumvent undesired product cleavage. Some approaches to suppress competitive reactions have been developed in our group, namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free micro-aqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems. Finally, for the first time, zymogens, which are known as catalytically inactive precursors of proteases, could be used as biocatalysts for practically irreversible peptide bond formation.

Asunto(s)

RESUMEN

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.

RESUMEN

The reverse action of hydrolases provides an attractive alternative to the chemical synthesis of peptides, oligosaccharides and oligonucleotides. Freezing the reaction mixture has proved to suppress competitive reactions in enzyme-catalysed peptide synthesis. After a short discussion of the influence of freezing on enzyme-catalysed reactions the current manuscript gives an overview of protease-catalysed peptide synthesis and the possible reasons of the yield-enhancing effect of freezing. The application of glycosidases and ribonucleases for synthetic purposes in frozen reaction mixtures is summarized.

RESUMEN

Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C (α,α) -disubstituted amino acids.α-Trifluoromethyl substituted amino acids, a subclass of C (α,α) -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containingα-TFM amino acids towards proteolysis byα-chymotrypsin. Furthermore, configurational effects ofα-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence ofα-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.

RESUMEN

The capability of the cysteine proteases ficin, papain and clostripain to form peptide bonds in frozen aqueous solutions was investigated. Freezing the reaction mixture resulted in increased peptide yields in kinetically controlled coupling of Bz-Arg-OEt with various amino acid amides and dipeptides. Under these conditions, peptide yields increased up to 70% depending on the enzyme and the amino component used. Enzyme-catalysed peptide syntheses were carried out under optimized reaction conditions (temperature, amino component concentration and pH before freezing) using the condensation of Bz-Arg-OEt and H-Leu-NH2 as a model reaction.

Asunto(s)

RESUMEN

Thermolysin-catalyzed (EC 3.4.24.4) and chymotrypsin-catalyzed (EC 3.4.21.1) peptide synthesis reactions were accomplished without any organic solvent in the presence of low amounts of water under sonication and fluidization. The systems used are considered to be microaqueous solvent-free ones. The influence of several reaction parameters, such as time, the amount of enzyme, the amount of water in free form or bound as hydration water, and the N/C component ratio, on the vield of the thermolysin-catalyzed synthesis of Z-Phe-Leu-NH(2) (up to 87% yield) was investigated in a sonicated system. Besides Z-Phe-Leu-NH(2), the tripeptide derivatives Ac-Xaa-Trp-Leu-NH(2), (Xaa = Gly, Ala) were also obtained in good yields of 79 and 71% respectively. In the latter case, no hydrolytic side reactions were observed. Using a fluidized-bed reactor, chymotrypsin- and thermolysin-catalyzed syntheses of N-protected di- and tripeptide amides could be perfromed with yields in the range of 10 to 40%. (c) 1995 John Wiley & Sons, Inc.

RESUMEN

Methodology for the synthesis and incorporation ofα-trifluoromethyl substituted amino acids into N- and C-terminal position of peptides is described. The incorporation ofα-trifluoromethyl substituted amino acids into strategical positions of peptides enhances proteolytic stability and lipophilicity. Furthermore, it improves transport rates in vivo and permeability through certain body barriers.