Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester to form N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z X Asp X Phe X OMe), the precursor to the sweet-tasting compound L-aspartyl-L-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-L-glycyl-L-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z X Asp X Phe X OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.     

Effect of the ΔPhe residue configuration on a didehydropeptides conformation: A combined CD and NMR study

Conformations of two pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐OMe ( Z‐ OMe ), Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐OMe ( E‐ OMe ), Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐p‐NA ( Z‐p‐ NA ), and Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐p‐NA ( E‐p‐ NA ) were compared on the basis of CD and NMR studies in MeOH, trifluoroethanol (TFE), MeCN, chloroform, and dimethylsulfoxide (DMSO). The CD results were used as the additional input data for the NMR‐based determination of the detailed solution conformations of the peptides. It was found that E‐ OMe is unordered and Z‐ OMe , Z‐p‐ NA , and E‐p‐ NA adopt the β‐turn conformation. There are two overlapping β‐turns in each of those peptides: type II and type III′ in Z‐ OMe and Z‐p‐ NA , and two type III in E‐p‐ NA . The ordered structure‐inducing properties of Δ^ZPhe and Δ^EPhe in the peptides studied depend on the C‐terminal blocking group. In methyl esters, the Δ^ZPhe residue is a strong inducer of ordered conformations whereas the Δ^EPhe one has no such properties. In p‐nitroanilides, both isomers of ΔPhe cause the peptides to adopt ordered structures to a similar extent. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1055–1064, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cerebral proteinases in the growing rat

—The proteolytic activity of brain homogenates obtained from 1-, 5-, 14-, 60-, 150-, and 300-day-old rats was assayed with urea-denatured haemoglobin and casein, endogenous tissue proteins, Nα-benzoyl-dl-arginine 2-naphtylamide (BANA), N^α-benzoyl-dl-arginine methyl ester (BAME), N^α-toluene p-sulphonyl-dl-arginine methyl ester (TAME), N^α-benzoyl-dl-phenylalanine 2-naphthyl ester (BPANE), and N^α-acetyl-dl-tyrosine ethyl ester (ATEE) as substrates. Several peaks of activity were detected with all these substrates in different pH ranges. Activity was highest with protein substrates at pH 3·0-4·0, with smaller peaks of activity at pH 5·5-6·5 and 8·0-9·0. At pH 3·0 the activity with trypsin substrates, viz. BANA, BAME and TAME, was also relatively high, but much less with chymotrypsin substrates, ATEE or BPANE. With BAME, TAME, BPANE and ATEE the hydrolysis rate was highest at neutral or slightly alkaline pH. During postnatal development the hydrolysis of protein substrates increased three-fold at pH 3·0 and about two-fold at pH 6·5 and 8·5. The rate of hydrolysis of BANA, BAME and TAME generally increased during the first 2 postnatal weeks and thereafter decreased, whereas no marked increase in the rate of hydrolysis of BPANE and ATEE occurred until the age of about 2 weeks. The results were less consistent with synthetic substrates than with protein substrates, indicating the existence of non-uniform alterations during development in the activity of the individual hydrolytic enzymes participating in the breakdown of brain proteins.     

Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k_cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K_m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Toward a synthesis of Clostridium butyricum apoferredoxin: Two tetradecapeptides comprising half the sequence (residues 7–20 and 21–34)

The two protected tetradecapeptides Z·Ser·Cys[Bzl(OMe)]·Val·Ser·Cys[Bzl(OMe]·Gly·Ala·Cys[Bzl(OMe)]·Ala·Gly·Glu(OBu^t)· Cys[Bzl(OMe)]·Pro·Val·NH·NH·Boc and Z·Ser·Ala·Ile·Thr·Gln·Gly·Asp(OBu^t)·Thr(Bu^t)·Gln·Phe·Val·Ile·Asp(OBu^t)·Ala·NH·NH·Boc, corresponding to residues 7–20 and 21–34 in the amino acid sequence of Clostridium butyricum apoferredoxin have been synthesized as a first stage in a total synthesis of the apoferredoxin. The former peptide has been deprotected to the tetra-thiol peptide H·Ser·Cys·Val·Ser·Cys·Gly·Ala·Cys·Ala·Gly·Glu·Cys·Pro·Val·NH·NH₂, and two tri-thiol and three di-thiol peptide components of this have also been synthesized for iron-sulfur complexing studies.     

Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Total enzymatic synthesis of cholecystokinin CCK-5

Summary. This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH₂ of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt·HCl by -chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester.The other fragment H-Asp(OMe)-Phe-NH₂ resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH₂·HCl and thermolysin as catalyst, followed by catalytic hydrogenation.Finally PhAc-Gly-Trp-Met-Asp-Phe-NH₂ was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH₂ with -chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the -methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.     

A convenient,rapid method for the resolution of enantiomeric amino acids using chiral phases on stainless-steel capillary gas chromatographic columns

An improved method is described for the resolution of enantiomeric isopropyl esters of N-trifluoroacetyl-α-amino acids of nonbasic amino acids using N-docosanoyl-l-valyl-t-butylamide and N-octadecanoyl-l-valyl-l-valine cyclohexyl ester as mixed chiral phases on 150-ft stainless-steel capillary columns. Enantiomers of Ala, Val, Ile, Leu, Ser, Thr, Asp, Met, Glu, and Phe are resolved in 105 min. This method avoids the fractionation problems and high costs encountered with the diastereometric method and difficulties and costs encountered in loading and maintaining glass capillary columns. It is particularly useful for studies involving a large number of resolutions as in a study of the kinetics of racemization of amino acids.     

Phosphatidylcholine synthesis in adult Dirofilaria immitis females

Crude homogenates of adult Dirofilaria immitis females were able to incorporate choline into phosphatidylcholine (PC) and also were able to methylate phosphatidyl (N,N-dimethyl)-ethanolamine, using S-adenosylmethionine as the methyl donor, to form PC. The finding of choline phosphotransferase (EC 2·7·8·2) and phosphatidyl (N,N-dimethyl) ethanolamine methyltransferase activity in the paniculate (mainly microsomal) fraction of the homogenates provided further evidence that adult D. immitis females can synthesize PC by way of choline and cytidine 5'-diphosphocholine (Kennedy pathway) and also by way of S-adenosylmethionine-mediated sequential methylation of phosphatidylethanolamine (Bremer-Greenberg pathway).     

Peptide Synthesis in Organic Media with the Use of Subtilisin 72 Immobilized on a Poly(Vinyl Alcohol) Cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.     

Crystal structure of tRNA N2,N2-guanosine dimethyltransferase Trm1 from Pyrococcus horikoshii

Trm1 catalyzes a two-step reaction, leading to mono- and dimethylation of guanosine at position 26 in most eukaryotic and archaeal tRNAs. We report the crystal structures of Trm1 from Pyrococcus horikoshii liganded with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. The protein comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds, respectively. The methyl moiety of S-adenosyl-l-methionine points toward the invariant Phe27 and Phe140 within a narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the monomethylated tRNA•Trm1 complex prior to the second methylation. Moreover, the catalytic residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.     

Aspartame precursor synthesis in water miscible cosolvent catalysed by thermolysin

Summary The synthesis of the dipeptideN-benzyloxycarbonyl-L- aspartyl-phenylalanine methyl ester, aspartame precursor, catalysed by thermolysin in aqueous and aqueous methanolic solutions was studied. Thermolysin with concentration as low as 10 M in 25% methanol can catalyse the synthetic reaction. The optimum methanol compositions at 4°C and 37°C were 50% and 25% respectively where an increase in peptide yield of 85% was obtained for both conditions as compared to that in water.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-aspartic acid - L-Phe-OMe L-phenylalanine methyl ester - N-cbz-L-Asp-Phe-OMe N-benzyloxycarbonyl-L-aspartyl-phenylalanine methyl ester All the % of methanol is a volume % in water unless otherwise specified.     

Kinetic behavior of activation of thermolysin by normal alcohols

Summary This paper reports an insight into the kinetic mechanism of activation and then inhibition in a thermolysin - catalyzed peptide synthesis of dipeptide N -(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester in aqueous - organic one phase system containing n-alcohols as activator. The jump in catalytic efficiency - as an effect of alcoholic solvents and the kinetic mode of activation are discussed.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Resolution of DL-2-aminosuberic acid via protease-catalyzed ester hydrolysis

Summary Papain-catalyzed regioselective cleavage of-methyl ester in Z-DL-Asu(OMe)-OMe leads to Z-L-Asu(OMe)-OH and Z-D-Asu(OMe)-OMe. Subsequent saponifications yield Z-L-Asu-OH and Z-D-Asu-OH. The enzymatic-ester hydrolysis was also achieved by subtilisin BPN in organic solvent with low water content.Abbreviations Asu 2-aminosuberic acid - Z benzyloxycarbonyl - OMe methyl ester - DCHA dicyclohexylamine     

Synthesis,conformation, and activity of HCO-Met-ΔZ Leu-Phe-Ome,an active analogue of chemotactic N-formyltripeptides

In order to induce a β-turn conformation into the chemotactic linear tripeptide N-formyl-L -methionyl-L -leucyl-L -phenylalanine (fMLP), the new analogue N-formyl-L -methionyl-Δ^Zleucyl-L -phenylalanine methyl ester [ Δ^ZLeu]²f MLP-OMe ( 1 ) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-α,β-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1 . The x-ray analysis shows that 1 adopts in the crystal a type II β-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. ¹H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl₃ solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by f MLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity. © 1993 John Wiley & Sons, Inc.     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.