Biomedical subjects

H D Jakubke

Publications and source records attributed to H D Jakubke.

At least 19 recordsLinked to original sources

PubMed1999

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

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.

Binding Sites↗

PubMed1998

Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic 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.

Amino Acids, Diamino↗

PubMed1998

The specificity of prolyl endopeptidase from Flavobacterium meningoseptum: mapping the S' subsites by positional scanning via acyl transfer.

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.

Acylation↗

PubMed1998

Reverse catalysis of elastase from porcine pancreas in frozen aqueous systems.

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.

Amino Acids↗

PubMed1997

Converting trypsin to chymotrypsin: structural determinants of S1' specificity.

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.

Amino Acid Sequence↗

PubMed1997

Influence of freeze-concentration effect on proteinase-catalysed peptide synthesis in frozen aqueous systems.

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.

Catalysis↗

PubMed1997

Reverse action of ribonuclease T1 in frozen aqueous systems.

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.

Dinucleoside Phosphates↗

PubMed1997

Subsite specificity studies on the unusual cysteine protease clostripain: charged residues in the P3 position indicate a narrow subsite region.

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.

Acylation↗

PubMed1994

The specificity of clostripain from Clostridium histolyticum. Mapping the S' subsites via acyl transfer to amino acid amides and peptides.

The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using N alpha-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-Xaa-Ala-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P'1 position, low specificity for amino acid amides and Xaa-(Ala)3-Gly peptides was found, however, in the P'2 position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P'1 and P'2 positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P'1-P'3 increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of P'3Phe. Surprisingly, P'1Gly leads to effective P'-S' interactions. However, the opposite result was obtained for P'2Gly. The high efficiency for Gly-NH2 does not fit with the hydrophobicity structure/activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH2 with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P'1Pro and P'2Gly and a negative cooperativity between P'1Pro and P'2Phe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.

Acylation↗

PubMed1994

S'-subsite mapping of polyethylene glycol-modified alpha-chymotrypsin and alpha-chymotrypsin: a comparative study.

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.

Amino Acid Sequence↗

PubMed1994

Acyl transfer reactions catalyzed by native and modified alpha-chymotrypsin in acetonitrile with low water content.

The characterization of the S' subsite specificity of native and ethylated alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl donor, we investigated the partitioning of acyl-chymotrypsins between water and amino acid and peptide-derived nucleophiles. For the investigation of S'2 subsite specificity, a series of 19 dipeptides of the general structure Ala-Xaa (Xaa represents all natural amino acids except cysteine) were used. From the values of the apparent partition constants rho app, the order of preference for the P'2 position is estimated to be: positively charged > hydrophilic > or = hydrophobic > aromatic > Pro > negatively charged side chain. Concerning the S'1 specificity, the same preference is deduced based on the study with the series of amino acid amides and Xaa-Ala dipeptides. In contrast to the nucleophilic specificity of alpha-chymotrypsin in aqueous solutions, free dipeptides and hydrophilic amino acid derivatives as nucleophiles exhibit much higher reactivities for acyl transfer in acetonitrile. We have not observed a significant difference in nucleophilic specificity between native and ethylated chymotrypsin.

Acetonitriles↗

PubMed1994

Enzymatic approach to the synthesis of taurine-containing peptides.

Subtilisins (subtilopeptidase A, nagarse) and proteinase K were able to catalyze the synthesis of taurine-containing peptides from various N-acylated amino acid or peptide esters and nonprotected taurine. The synthesis was optimized using a model reaction between Boc-Tyr-OMe and taurine. The best results were obtained under strongly alkaline conditions in acetonitrile with low water content as the reaction medium. The choice of the base added to the reaction medium had a substantial effect on the product yield. A preparative synthesis of Tyr-Tau and Ala-Phe-Tau is described.

Amino Acid Sequence↗

PubMed1994

Kinetic characterization of affinity chromatography purified clostripain.

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.

Arginine↗

PubMed1994

Enzymatic peptide synthesis in frozen aqueous systems: influence of modified reaction conditions on the peptide yield.

The alpha-chymotrypsin (EC 3.4.21.1)-catalyzed reaction of Mal-Phe-OMe with H-Leu-NH2 has been studied under a range of reaction conditions, for example various cryogenic reagents for shock-freezing, addition of dimethyl sulfoxide (DMSO) and decreased reaction temperatures down to 213 K. It has been shown that the peptide yield is independent of the method of shock-freezing. The optimal reaction temperature was between 263 K and 248 K. Lower temperatures result in clearly retarded reactions. Addition of DMSO leads to decreasing peptide yields. It is certain that the peptide bond formation is catalyzed by the active enzyme, since unspecific protein surface catalysis gave no peptide yields at all.

Caseins↗

PubMed1993

Peptide synthesis by chymotrypsin in frozen solutions. Free amino acids as nucleophiles.

Nucleophilic efficiency of the free amino acids in chymotrypsin-catalyzed acyl transfer in ice at -18 degrees C using ethyl esters of N-maleyl-L-tyrosine and L-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln > Lys > Cit > Ala > Ala > Gly > Asn > Arg > Glu > Val > Orn > Asp with no peptide formed with His, Leu, Ile and Pro) for N-maleyl-L-tyrosine ethyl ester and Ser > Lys > Orn > Arg,Cit > Gln > Thr > Asn > Ala > Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for L-tyrosine ethyl ester.

Amino Acids↗

PubMed1992

Contributions to the S'-subsite specificity of papain.

The product ratio was analyzed for the papain-catalyzed acyl transfer from the specific acyl donor Mal-Phe-Ala-OEtCl to various nucleophilic amino components, ranging from amino acid amides to tripeptide amides. The data obtained are discussed in terms of binding specificity. From the structure-activity relationships for the S'1-P'1 interaction it follows that only three methyl(ene) groups can be accommodated in the S'1 subsite. Hydrophilic side chains are bound better to S'1 than indicated by their hydrophobicities. Negatively charged amino components are inefficient deacylating agents. However, there was no evidence for electrostatic contributions to the nucleophile binding. Amino components with bulky hydrophobic amino acid residues in the P'2 and in the P'3 position, respectively, are preferentially bound to Mal-Phe-Ala-papain. The results of this study can be applied to the planning of papain-catalyzed peptide synthesis reactions.

Acyltransferases↗

PubMed1991

Penicillin acylase-catalyzed protection and deprotection of amino groups as a promising approach in enzymatic peptide synthesis.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease-catalyzed peptide synthesis. Here the synthesis of leucine-enkephalin using enzymes for N-terminal amino group protection, peptide bond formation and deprotection is described.

Amino Acid Sequence↗

PubMed1991

The specificity of chymotrypsin. A statistical analysis of hydrolysis data.

From the literature we collected all available quantitative data on the chymotrypsin-catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(kcat/Km) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.

Amino Acid Sequence↗