Biomedical subjects

L Polgár

Publications and source records attributed to L Polgár.

At least 19 recordsLinked to original sources

PubMed2005

The catalytic triad of serine peptidases.

The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid. In the classic trypsin and subtilisin families this catalytic triad is composed of serine, histidine and aspartic acid residues and exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence is different. By now several new families have been discovered, in which the nucleophile-base-acid pattern is generally conserved, but the individual components can vary. The variations illustrate how different groups and different protein structures achieve the same reaction.

Amino Acids↗

PubMed2005

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity.

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.

Alternative Splicing↗

PubMed2002

The prolyl oligopeptidase family.

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed beta-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis.

Animals↗

PubMed2001

Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of picornain 3C.

Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.

3C Viral Proteases↗

PubMed2001

Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Amino Acid Substitution↗

PubMed2000

Characterization of the active site thiol group of rhinovirus 2A proteinase.

Picornains 2A are cysteine proteases of picornaviruses, a virus family containing several human and animal pathogens. The pH dependencies of the alkylations of picornain 2A of rhinovirus type 2 with iodoacetamide and iodoacetate show two reactive thiol forms, namely the free thiolate ion at high pH and an imidazole assisted thiol group at low pH. Kinetic deuterium isotope effects do not support general base catalysis by the imidazole group, but rather the existence of a catalytically competent thiolate-imidazolium ion-pair. The nature of the ion-pair differs from that of papain, the paradigm of cysteine proteases. The ion-pair is confined to the same, unusually narrow pH range in which the enzyme exhibits catalytic activity.

Binding Sites↗

PubMed2000

The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain.

Prolyl oligopeptidase, which is involved in memory disorders, is a member of a new family of serine peptidases. In addition to the peptidase domain, the enzyme contains a beta-propeller, which excludes large peptides from the active site. The enzyme is inhibited with thiol reagents, possibly by reacting with Cys-255 located close to the substrate binding site. This assumption was tested with the Cys-255 --> Thr, Cys-255 --> Ala, and Cys-255 --> Ser variants of prolyl oligopeptidase. In contrast to the wild type enzyme, the Cys-255 --> Thr variant was not inhibited with N-ethylmaleimide, indicating that Cys-255, of the 16 free cysteine residues, exclusively accounts for the enzyme inhibition. Unlike the wild type enzyme that showed a doubly bell-shaped pH rate profile, the modified enzyme displayed a single bell-shaped pH dependence with benzyloxycarbonyl-Gly-Pro-naphthylamide. It was the high pH form of the enzyme that virtually disappeared with all three enzyme variants. A substantial reduction was also observed in k(cat)/K(m) for the aminobenzoyl-Ser-Pro-Phe(NO(2))-Ala-OH substrate. The high pK(a) (9.77) of Cys-255 determined by titration with N-ethylmaleimide excluded the possibility that ionization of the thiol group was responsible for generation of the two active enzyme forms. The impaired activity of the enzyme variants could be rationalized in terms of weaker binding, which manifests itself in high K(m) for substrates and high K(i) for inhibitors, like benzyloxycarbonyl-Gly-Pro-OH and aminobenzoyl-Ser-d-Pro-Phe(NO(2))-Ala-OH. It was concluded that, besides selecting substrates by size, the beta-propeller domain containing Cys-255 remarkably contributed to catalysis of the peptidase domain.

Amino Acid Substitution↗

PubMed2000

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism.

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed beta-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.

Amino Acid Substitution↗

PubMed2000

Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease.

The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1). 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

Acrylamide↗

PubMed2000

Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family.

Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide. With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km. The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.

Amino Acid Sequence↗

PubMed1999

Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity.

Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, is involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of approximately 8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 degrees C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 degrees C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.

Animals↗

PubMed1998

Crystallization and preliminary X-ray analysis of porcine muscle prolyl oligopeptidase.

Prolyl oligopeptidase from pig muscle has been crystallized in complex with an inhibitor, using PEG 8000 and calcium acetate as precipitants. The crystals are orthorombic and the space group is P212121 with cell dimensions a = 111.8, b = 101.8, c = 72.4 A. The asymmetric unit contains a single chain of prolyl oligopeptidase, corresponding to a specific volume of 2.55 A3 Da-1 and a solvent content of 52%. The observed diffraction pattern extends to 2.3 A resolution and the native crystals are well suited for structural analysis by X-ray diffraction methods.

Animals↗

PubMed1998

Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis.

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

Amino Acid Sequence↗

PubMed1998

Oligopeptidase B: cloning and probing stability under nonequilibrium conditions.

Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions. To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1-Blue, and purified essentially in two chromatographic steps. The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, deltaG0. Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat. Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements. A static method, intrinsic fluorescence vs. pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5. The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant changes. Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method. Acid denaturation at pH 3 was markedly retarded by 1 M NaCl. Against heat inactivation the enzyme was also considerably protected in the presence of salt, and the higher enthalpy and entropy of activation suggested the importance of hydration in the stabilization. The kinetics of unfolding followed single-exponential decay under strongly denaturing conditions (high urea concentration or high temperature), but deviated from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH. The results indicate that under harsher denaturing conditions there is a single rate-limiting step in unfolding, whereas under milder conditions partly unfolded intermediates are populated.

Cloning, Molecular↗

PubMed1997

Low barrier hydrogen bond is absent in the catalytic triads in the ground state but Is present in a transition-state complex in the prolyl oligopeptidase family of serine proteases.

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

Animals↗

PubMed1997

A potential processing enzyme in prokaryotes: oligopeptidase B, a new type of serine peptidase.

Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products. Such enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes. Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidase. The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates. The kcat/K(m) is extremely high, 63 microM-1 s-1, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4-methylcoumaryl)amide. The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s). This effect is abolished at 1 M NaCl. In addition, high ionic strength inhibits the reaction considerably by increasing K(m), which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups. In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis. The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms.

Bacterial Proteins↗

PubMed1996

Rate-determining steps in HIV-1 protease catalysis. The hydrolysis of the most specific substrate.

The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2' position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO2)-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2' and P3' sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (kcat/Km = 2.1 x 10(7) M-1 s-1) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating that, independent of the salt concentration, the rate-limiting step of the catalysis is a chemical process rather than a physical one. The reaction also has positive entropy of activation. On the other hand, the rate-limiting step for substrate QR changes with increasing salt concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5-fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The Km value is much lower for the substrate ER (0.8 microM) than for substrate QR (15 microM), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the strong binding appears to be achieved by the unionized form of Glu in P2', as follows from the remarkably different pH-rate profiles for substrates QR and ER. The effective binding elicited by the glutamic acid may be utilized in designing inhibitors for therapeutic purposes.

HIV Protease↗

PubMed1996

Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration.

The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I) (Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J. M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C., Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G. (1994) Biochemistry 33, 9405-9413), designed to better resist autolysis. Monitoring the intrinsic fluorescence of the two enzymes during urea-mediated denaturation has shown that at high NaCl concentration, both the conformational stability ( DeltaG0) and the transition midpoint (D1/2) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far- and near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a two-state equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.

Amino Acid Sequence↗