Analysis of Toxoplasma gondii clonal type-specific antibody reactions in experimentally infected turkeys and chickens.

Due to their ground-feeding behaviour, free-ranging chickens and turkeys are exposed to oocysts and are good indicators of the presence of Toxoplasma gondii in the environment. In addition, poultry may become infected by ingestion of tissues of infected intermediate hosts such as small rodents. Free-ranging poultry are considered an important source of T. gondii infection in humans, especially in developing countries. Knowledge on T. gondii genotypes in infected animals and humans is important for understanding the epidemiology of T. gondii infections. The aim of the present study was to analyse the ability of experimentally infected turkeys and chickens to develop a T. gondii clonal type-specific antibody response (IgY) after i.v. inoculation with tachyzoites of three T. gondii clonal lineages, types I, II and III. A peptide microarray displaying a panel of 101 different synthetic peptides was used for serotyping. Peptide sequences were derived from polymorphic regions of 16 T. gondii proteins (GRA1, GRA3-7, SAG1, SAG2A, SAG3, SAG4, SRS1, SRS2, ROP1, NTPase I and NTPase III and BSR4). The array was probed with 120 sera from experimentally infected chickens and turkeys inoculated with different doses of T. gondii tachyzoites (104, 103 and 102) collected from isolates representative for T. gondii clonal types I (RH), II (ME49) or III (NED) and uninfected controls. After screening of the peptides with reference sera from chickens and turkeys, and evaluation of data by Receiver Operating Characteristics analysis, 41 and 40 peptides were identified that appeared suitable to detect type-specific reactions with sera collected at 2, 5, 7 and 9 weeks p.i. Selected peptides allowed the identification of T. gondii clonal types, until 9 week p.i., which the chickens or turkeys had been inoculated with. At 9 weeks p.i., a high proportion of the experimentally infected chickens (67% (12/18)) and turkeys (61% (11/18)) no longer reacted with the selected peptides. Serotyping of the infection in individual chickens or turkeys was only possible when the whole peptide panel was applied. Clonal type-specific antibody responses were dynamic in both poultry species and depended on the individual animal and the time after infection.

Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase.

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.

The speed limit for protein folding measured by triplet-triplet energy transfer.

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N = 3) with a time constant (tau = 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.

Mapping the stereospecificity of peptidyl prolyl cis/trans isomerases.

The stereospecificity of peptidyl prolyl cis/trans isomerases (PPIases) was studied using tetrapeptide substrate analogs in which one amino acid residue was replaced by the cognate D-amino acid in various positions of the peptide chain. Reversed stereocenters around proline markedly increased the rate of the spontaneous trans to cis isomerization of the prolyl bond whereas cis to trans isomerizations were less sensitive. PPIases like human cyclophilin18, human FKBP12, Escherichia coli parvulin10 and the PPIase domain of E. coli trigger factor exhibited stereoselectivity demanding at the P1 to P2' position of the substrate chain. The discriminating factor for stereoselectivity was the lack of formation of the Michaelis complexes of the diastereomeric substrates. However, D-alanine at the P1 position preserved considerable affinity to the active site, and largely prevented activation of the catalytic machinery for all PPIases investigated.

Side-chain effects on peptidyl-prolyl cis/trans isomerisation.

Peptidyl-prolyl cis/trans isomerisation has been frequently found as a rate limiting step in the folding of proteins. In order to determine whether the nature of the amino acid preceding proline controls the probability of cis prolyl bonds in native proteins, systematic studies on the thermodynamics and kinetics of the prolyl isomerisation in the pentapeptide series Ac-Ala-Xaa-Pro-Ala-Lys-NH2 were performed. All proteinogenic amino acids were substituted in the position preceding proline. When measured by 1H-NMR and CD spectroscopy both isomers proved to be devoid of ordered structure in the whole series of the oligopeptides in aqueous solution. Thus, isomerization rates and cis/trans ratios calculated from solvent jump and 1H-NMR magnetisation transfer experiments exclusively reflect the side-chain effects of the Xaa position in the peptide series. There is a rough correlation between the cis content in the oligopeptides and the propensity of Xaa-Pro cis prolyl bonds in proteins. This correlation suggests that the prolyl bond conformation is mainly determined by local effects in proteins. The rate constants kc-->t of pentapeptides containing unionised amino acids preceding proline range from 3.2 x 10(-3) s-1 (Xaa = Ala) to 0.5 x 10(-3) s-1 (Xaa = Trp) at 4 degrees C. Proline clustering led to an isomerisation cycle indicating considerable influence on the isomerisation rates of the peptide bond conformations flanking the rotating bond. Both tyrosine and histidine specifically reduce isomerisation rates severalfold by deprotonation of their respective side-chains.

Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition.

Proline residues provide a backbone switch in a polypeptide chain, which is controlled by the cis/trans isomerization about the peptidyl-prolyl bond. Phosphorylation of serine- and threonine-proline motifs has been shown to be a critical regulatory event for many proteins. The biological significance of these motifs has been further highlighted by the discovery of a novel and essential peptidyl-prolyl cis/trans isomerase Pin1. Pin1 is required for progression through mitosis via catalyzing the isomerization of phosphorylated Ser/Thr-Pro motifs specifically present in mitosis-specific phosphoproteins. However, little is known whether the phosphorylation regulates the conformational switch of the Ser/Thr-Pro bonds. Here, we report the synthesis and conformational characterization of a series of peptides that contain the phosphorylated or nonphosphorylated Ser/Thr-Pro motifs. Phosphorylation affected the rate of the cis to trans isomerization of the Thr/Ser-Pro bonds. As determined by a protease-coupled assay, the isomerization rate of phosphorylated Thr-Pro bond was found to be 8-fold slower than that of the nonphosphorylated analogue. Furthermore, studies of the pH dependence of the isomerization of the phosphopeptides reveal that both cis content and the rate constant of prolyl cis to trans isomerization are lower for the dianionic state of the phosphothreonine-containing peptides. These effects of phosphorylation are specific for phosphorylated Ser/Thr since neither phosphorylated Tyr nor glutamic acid was able to affect the prolyl isomerization. Finally, our experiments provide evidence that effective catalysis of cis/trans isomerization of phosphorylated Ser/Thr-Pro bonds by Pin1 is specific to the dianionic form of the substrate. Thus, our results demonstrate that protein phosphorylation specifically regulates the backbone dynamics of the Ser/Thr-Pro motifs and that Pin1 specifically isomerizes the certain conformation of the phosphorylated Ser/Thr-Pro motifs.

Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1.

This study was initiated to determine whether the intestinal H+/peptide symporter PEPT1 differentiates between the peptide bond conformers of substrates. We synthesized a modified dipeptide where the peptide bond is replaced by the isosteric thioxo peptide bond. The Ala-Pro derivative Ala-psi[CS-N]-Pro exists as a mixture of cis and trans conformation in aqueous solution and is characterized by a low cis/trans isomerization rate. The compound was recognized by PEPT1 with high affinity. The Ki value of Ala-psi[CS-N]-Pro for the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2 cells was 0.30 +/- 0.02 mM, determined in solution with 96% trans conformation. In contrast, the Ki value was 0.51 +/- 0.02 mM when uptake media with 62% trans conformer were used. We conclude that only the trans conformer interacts with the transport system. From our data, a significant affinity of the cis conformer at PEPT1 cannot be derived. In a second approach, conformer-specific uptake of Ala-psi[CS-N]-Pro was studied by analyzing the intracellular content of Caco-2 cells following transport as well as the composition of the extracellular medium using capillary electrophoresis. The percentage of trans conformer that was 62% in the uptake medium increased to 92% inside the cells. This is the first direct evidence that an H+/peptide cotransport system selectively binds and transports the trans conformer of a peptide derivative.

Intramolecular assistance of cis/trans isomerization of the histidine-proline moiety.

Peptidyl-prolyl cis/trans isomerization is a slow conformational interconversion in the polypeptide backbone that is frequently rate-limiting in refolding of proteins and is thought to play a role in cellular restructuring of proteins. In order to probe the influence of positively charged amino acids located in sequence segments adjacent to proline, the rotational barriers of Arg-Pro- and His-Pro-containing peptides were determined by isomer-specific proteolysis and dynamic NMR spectroscopy for Suc-Ala-His-Pro-Phe-NH-Np, Ac-Ala-Arg-Pro-Ala-Lys-NH2, Ac-Ala-His-Pro-Ala-Lys-NH2, angiotensin III, thyrotropin-releasing hormone (TRH), and [His(3-Me)2]TRH in aqueous solution. In contrast to the guanidinium group of arginine, the protonated side chain of histidine preceding proline led to an acceleration of the prolyl isomerization up to 10-fold relative to the unprotonated state. Both arginine and histidine residues succeeding proline in an amino acid sequence proved to be ineffective. Under basic and acidic conditions the kinetic solvent deuterium isotope effects Kc-->tH20/Kc-->tD20 for angiotensin III were 1.0 +/- 0.1 and 2.0 +/- 0.1, respectively. The results are interpreted in terms of intramolecular general acid catalysis of prolyl bond rotation by the imidazolium group that is without precedent in intermolecular catalysis.

A protease-free assay for peptidyl prolyl cis/trans isomerases using standard peptide substrates.

Peptidyl prolyl cis/trans isomerases (PPIases) are ubiquitous and abundant enzymes catalyzing peptide bond cis/trans isomerization adjacent to proline in peptides and proteins. An uncoupled protease-free assay of PPIase activity has been developed using the standard tetrapeptide substrates of the proteolytically coupled test system. Differences in the UV/vis absorption spectra of cis and trans conformations of Suc-Ala-Xaa-Pro-Phe-(Y-) anilide (Xaa = Ala, Leu, Phe; Y = 4-nitro, 2,4-difluoro) were exploited to monitor the time course of the cis/trans isomerization subsequent to a solvent jump from 0.47 M LiCl/trifluoroethanol into aqueous solution. The utility of the assay has been demonstrated by the determination of the Michaelis-Menten constants of cytosolic cyclophilin (Cyp18) and of the proteolytically sensitive FK506-binding protein-like PPIase SlyD from Escherichia coli. Furthermore, similar inhibition constants were estimated for the reversible inhibition of human Cyp18 by cyclosporin A (CsA) with both the proteolytically coupled and the novel uncoupled PPIase assay.

The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides.

Peptide-4-nitroanilides can be quickly synthesised using an Fmoc-based approach on 2-chlorotritylchloride resin. Preformed building blocks Fmoc-Xaa-NH-Np (Xaa = Cit, Cys, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp) can be attached via side chain to the 2-chlorotritylchloride linker of the resin. N-terminal elongation yields the respective peptide-4-nitroanilides after detachment from the solid support. We synthesised a set of tetrapeptide-4-nitroanilides with the general structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Asp, Cit, Cys, Glu, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp). Even peptidyl-arginine-4-nitroanilides are available by a slightly modified procedure. First, the appropriate ornithine-containing peptide was synthesised. After detachment of the peptide from the resin the side-chain primary amino group was transformed to the guanidino function of arginine using 1-guanyl-3,5-dimethylpyrazole. A further application of this method is the convenient synthesis of phosphorylated peptide-4-nitroanilides. Five phosphopeptides with the general structure Ac-Ala-Xaa(PO3H2)-Pro-Yaa-NH-Np (Xaa = Ser, Thr, Tyr; Yaa = Tyr, Lys) and their nonphosphorylated analogues were prepared. Global phosphorylation was carried out on the resin-bound peptides using dibenzyl-N, N-diisopropyl-phosphoramidite/tetrazole followed by oxidation with tert-butyl hydroperoxide.

Conformational state of a 25-mer peptide from the cyclophilin-binding loop of the HIV type 1 capsid protein.

Recently a 25-residue part of Gag polyprotein from HIV type 1 (HIV-1) was reported to bind to the cytosolic 18 kDa cyclophilin (Cyp18) with an IC50 value of 180 microM. This peptide corresponds to the Cyp18-binding domain of HIV-1 Gag. A replacement of Gly with Ala in the cyclophilin-binding loop of HIV-1 Gag polyprotein results in the prevention of the packaging of Cyp18 into virions. We found only two conformers of this peptide among 16 possible expected conformers, owing to cis/trans isomerization of four peptidyl-prolyl bonds. Although this finding implicates the existence of a stabilizing structure, we were not able to detect secondary structure formation by 1H-NMR and CD spectroscopy. We characterized the peptide as a substrate for Cyp18 by two-dimensional exchange 1H-NMR spectroscopy. Surprisingly, we found similar binding characteristics for a peptide corresponding to 25-mer peptide containing the above-mentioned Gly to Ala substitution.

Probing substrate backbone function in prolyl oligopeptidase catalysis--large positional effects of peptide bond monothioxylation.

Site-specific effects on the catalytic activity of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond. Two series of tetrapeptide-4-nitroanilides, Ala-Gly-Pro-Phe-NH-Np and Ala-Ala-Pro-Phe-NH-Np, along with all possible monothioxylated derivatives, were synthesised and k(cat) and Km values were determined for proteolytic cleavage at the Pro-Phe bond. Regardless of either Gly or Ala in the P2 subsite, tetrapeptides were rendered uncleavable by thioxylation at the Pro-Phe linkage. As a result, Ala-Xaa-Pro-psi[CS-NH]-Phe-NH-Np (Xaa = Gly or Ala) displayed competitive inhibition with Ki-values of 12 microM and 44 microM, respectively. Furthermore, in controlling proteolytic susceptibility of the substrates, cooperation of the P3-P2 thioxylation site and the side chain at the P2 subsite was obtained. Thioxylation at this position enhanced k(cat)/Km fivefold in the Gly series, but led to a 1.7-fold decrease in the Ala series of substrates. With respect to the Xaa-Pro peptide bond, all of the substrates underwent cis/trans isomerisation, thus presenting two stable conformers to the protease. However, the magnitudes of the isomerisation constants suggested that neither isomerisation rates nor cis/trans equilibria can explain the effect of thioxylation on the steady-state constants of proteolysis.

Cyclophilin A complexed with a fragment of HIV-1 gag protein: insights into HIV-1 infectious activity.

BACKGROUND: Cyclophilin A (CyPA), a receptor of the immunosuppressive drug cyclosporin A, catalyzes the cis-trans isomerization of peptidyl-prolyl bonds and is required for the infectious activity of human immunodeficiency virus type 1 (HIV-1). The crystal structure of CyPA complexed with a fragment of the HIV-1 gag protein should provide insights into the nature of CyPA-gag interactions and may suggest a role for CyPA in HIV-1 infectious activity. RESULTS: The crystal structure of CyPA complexed with a 25 amino acid peptide of HIV-1 gag capsid protein (25-mer) was determined and refined to an R factor of 0.195 at 1.8 A resolution. The sequence Ala88-Gly89-Pro90-Ile91 of the gag fragment is the major portion to bind to the active site of CyPA. Two residues of the 25-mer (Pro90-Ile91) bind to CyPA in a similar manner to two residues (Pro-Phe) of the CyPA substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). However, the N-terminus of the 25-mer (Ala88-Gly89) exhibits a different hydrogen-bonding pattern and molecular conformation than AAPF. The peptidyl-prolyl bond between Gly89 and Pro90 of the 25-mer has a trans conformation, in contrast to the cis conformation observed in other known CyPA-peptide complexes. The residue preceding proline, Gly89, has an unfavorable backbone conformation usually only adopted by glycine. CONCLUSIONS: The unfavorable backbone conformation of Gly89 of the gag 25-mer fragment suggests that binding between HIV-1 gag protein and CyPA requires a special sequence, Gly-Pro. Thus, in HIV-1 infectivity, CyPA is likely to function as a chaperone, rather than as a cis-trans isomerase. However, the observation of similarities between the C termini of the 25-mer and the substrate AAPF means that the involvement of the cis-trans isomerase activity of CyPA cannot be completely ruled out.

Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism.

Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.

Solid-phase synthesis of peptide-4-nitroanilides.

A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides and other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing the side-chain carboxyl groups for transient anchoring to the resin. Suitable synthons for this method, Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptides of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Glu/Asp, Gln) were synthesized and were shown to be substrates for the protease subtilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans-isomerases (PPIases E.C. 5.2.1.8.). The method was extended to amino acids possessing a side chain missing an anchor for binding to the matrix. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptide derivative Fmoc-Glu-Phe-NH-Np with the carboxyl group to Rink amide resin using standard SPPS procedures. Additionally this procedure allowed us the preparation of peptidyl-arylamides, utilizing the commercial available Fmoc-Glu-OAll as building block. A mixture of pentapeptide-4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np was synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the protease subtilisin Carlsberg. It could be shown that peptides with the hydrophobic amino acids Phe, Tyr, Leu and Val in the varied P3-position were most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed.

Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.

Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.

Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique.

Often protein folding reactions show complex kinetics, because multiple unfolded species are present, which refold simultaneously. After conformational unfolding, these species are formed by the slow cis/trans equilibrations at Xaa-Pro peptide bonds. To dissect the roles of individual prolines for unfolding and refolding, we used ribonuclease T1, a protein with two cis prolyl peptide bonds, preceding Pro39 and Pro55, and two variants with substitutions at these positions. A stopped-flow double-mixing technique was employed (i) to measure the rates of the individual prolyl isomerizations in the unfolded proteins and (ii) to study the refolding of transient species that are not well populated at equilibrium. In particular, the elusive species with correct prolyl isomers could be produced by short unfolding pulses, and its refolding kinetics could be measured. The two isomerizations in unfolded ribonuclease T1 could be assigned to Pro39 and Pro55, because they occurred with almost identical rates in the wild-type protein, in the single-cis proline variants, and in tetrapeptide-4-nitroanilides, which contained prolines in the same sequential context at Pro39 and Pro55 or ribonuclease T1. The direct refolding reaction of the unfolded molecules with correct prolyl isomers shows a time constant of 180 ms (at 25 degrees C, pH 4.6). This reaction is almost unaffected by the proline substitutions. It depends nonlinearly on temperature with a maximum near 25 degrees C, which suggest that the activated state for this reaction resembles the native rather than the unfolded state in heat capacity. The formation of a transient intermediate with incorrect prolyl isomers could be studied as well. Surprisingly, this reaction is only about 5-fold slower than direct folding, and it is also accompanied by a strong decrease in the apparent heat capacity.

A model of the active site of dipeptidyl peptidase IV predicted by comparative molecular field analysis and molecular modelling simulations.

A molecular model of the active site of the serine protease dipeptidyl peptidase IV (DPP IV or CD26) has been developed on the basis of comparative molecular field analysis (CoMFA) of competitive inhibitors and by force field calculations. By application of CoMFA experimentally obtained inhibition constants Ki have been successfully predicted. The resulting steric and electrostatic coefficients of CoMFA were used for the development of the molecular model. The main assumptions of the model are the recognition of substrates or inhibitors by the side chains of a tyrosine (S1-position) and a tryptophan residue (S2-position). The model helps us to understand a multitude of experimental data regarding the substrate specificity of this enzyme as well as results obtained by genetic engineering experiments by other authors. General conclusions concerning a new family of serine proteases are drawn and discussed.

Inhibition of peptidyl-prolyl cis/trans isomerase activity by substrate analog structures: thioxo tetrapeptide-4-nitroanilides.

The ubiquitous cyclophilins belong to peptidyl-prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). They are able to catalyze the cis/trans isomerization about peptidyl-prolyl amide bonds. The mode of action of human cytosolic cyclophilin (Cyp18cy) has been studied on substrate analog tetrapeptide-4-nitroanilides containing the thioxo peptidyl-prolyl bond. Five peptides of the general structure Ala-Xaa-psi [CS-N]-Pro-Phe-NH-Np (Xaa = Gly, Ala, (S)-2-aminobutyric acid, Phe, and Leu) containing the thioxo peptidyl-prolyl bond were synthesized. The kcat values for the chymotryptic cleavage of 4-nitroanilide bond of the thioxo tetrapeptide-4-nitroanilides ranged from 1.7 to 9.0 s-1 and were sufficiently high to analyze the conformational equilibria by isomer-specific proteolysis. The rate constants of the cis/trans isomerization of the thioxo peptidyl-prolyl bond were found to be 25-100-fold lower due to the O/S substitution. Cyp18cy binds both thioxo peptides and oxo peptides in similar manner in the active center but cannot utilize the sulfur analogs as substrates. Instead, competitive inhibition occurs, which was further characterized for Ala-Gly-psi[CS-N]-Pro-Phe-NH-Np. The inhibition was nearly independent of the pH value in the range of pH 4.5-9, exhibiting apparent Ki values ranging from 200 to 600 microM. In comparison to Ala-Gly-trans-psi[CS-N]-Pro-Phe-NH-Np, the cis thioxo peptide Ala-Gly-cis-psi[CS-N]-Pro-Phe-NH-Np was found to possess an approximately 30-fold higher affinity for the active site of the enzyme. Thus, in the presence of stoichiometric amounts of Cyp18cy, the total amount of Ala-Leu-cis-psi[CS-N]-Pro-Phe-NH-Np in solution, detectable by isomer-specific proteolysis, was considerably enhanced.