András Patthy

Trypsin Complexed with α1-Proteinase Inhibitor Has an Increased Structural Flexibility

Mutant rat trypsin Asp189Ser was prepared and complexed with highly purified human α 1‐proteinase inhibitor. The complex formed was purified to homogeneity and studied by N‐terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with α 1‐proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg117‐Val118 one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase‐substrate and serine proteinase‐serpin reactions are discussed.

Effect of bacitracin on the biodegradation of β-endorphin

Abstract β-endorphin was incubated with rat brain homogenate, and the amino acids released were measured by amino acid analysis. Phe, Leu, Tyr, and Lys were liberated in the greatest amount indicating that the cleavage of Leu77-Phe78 and some Lys-X peptide bonds with endopeptidases followed by the removal of the terminal residues by exopeptidases are the main routes of β-endorphin degradation in the brain. Bacitracin considerably reduced the amino acid release from β-endorphin incubated with rat brain homogenate, and its action is suggested to be due to the inhibition of brain amino- and carboxypeptidases. Bacitracin also potentiated and prolonged the in vivo analgesic activity of β-endorphin.

Studies on the primary structure of human β-lipotropic hormone

The isolation and some characteristics of a lipolytic peptide from human pituitaries were described in our previous paper [ 1 ] . On the basis of chemical and biological studies this peptide was assumed to be a species homologue of the fl-lipotropic hormone @-LPH) discovered by Li and coworkers [ 21. Meanwhile the isolation and complete sequence of porcine @-LPH has been reported from our laboratory [3,4] , Recently, we have extended our structural studies to the human P-LPH. In this paper we present some results of this work.

Solution Structure of the Major alpha-amylase Inhibitor of the crop plant Amaranth

α-Amylase inhibitor (AAI), a 32-residue miniprotein from the Mexican crop plant amaranth (Amaranthus hypochondriacus), is the smallest known α-amylase inhibitor and is specific for insect α-amylases (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R., and Pongor, S. (1994) J. Biol. Chem. 269, 23675–23680). Its disulfide topology was confirmed by Edman degradation, and its three-dimensional solution structure was determined by two-dimensional 1H NMR spectroscopy at 500 MHz. Structural constraints (consisting of 348 nuclear Overhauser effect interproton distances, 8 backbone dihedral constraints, and 9 disulfide distance constraints) were used as an input to the X-PLOR program for simulated annealing and energy minimization calculations. The final set of 10 structures had a mean pairwise root mean square deviation of 0.32 Å for the backbone atoms and 1.04 Å for all heavy atoms. The structure of AAI consists of a short triple-stranded β-sheet stabilized by three disulfide bonds, forming a typical knottin or inhibitor cystine knot fold found in miniproteins, which binds various macromolecular ligands. When the first intercystine segment of AAI (sequence IPKWNR) was inserted into a homologous position of the spider toxin Huwentoxin I, the resulting chimera showed a significant inhibitory activity, suggesting that this segment takes part in enzyme binding.

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.

ABSTRACT Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (∼74, ∼55, ∼54, and ∼37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.

Probing conformational plasticity of the activation domain of trypsin: The role of glycine hinges

Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.

Enkephalin analogs containing amino sulfonic acid and amino phosphonic acid residues at position 5

Enkephalins, the endogenous opioid peptides: Tyr-Gly-Gly-Phe-Met and -Leu [ 11, have been known to show much higher agonist potency in the mouse vas deferens preparation (MVD) than in the guinea-pig ileum assay system (GPI) [2-61. The high MVD/GPI potency ratio is characteristic of enkephalins and of their synthetic analogs unless the pentapeptides are terminated by an amide function. For instance, potency ratios calculated for Met-enkephalin, its D-Ala2 analog and [D-Ala2, Met’]-enkeph~~ide were 9.0,8.4 and 2.0, respectively [2]. The MVDlGPI ratio for &endorphin, the 3 l-residue opioid peptide of the pituitary, is -1.2 and for nonpeptide narcotics, e.g., normorphine and morphine even smaller [2,4-61. Accordingly, the acidic COOH group at the C-terminus is essential for the ‘enkephalinoid’ character of pentapeptides. Starting from this consideration we synthesized some analogs of norleucine (Nle)-enkephahn in which the terminal COOH group is replaced by SOaH and POsH, group, respectively, i.e., Me’ is ~bstituted by a-amino~nt~e-s~fonic acid (NleS) and ff-amino-pentanephosphonic acid (MeP), respectively. The dissociation constant of a sulfonic acid or a phosphonic acid is known to be higher than that of the corresponding carboxylic acid. For instance, pKL values of taurine and p&nine are 1.5 and 3.6, respectively [7]. Similarly, pKH of glycine is 2.34 while the two pKH values of amino-methane-phosphonic acid are 1.85 and 5.35, respectively [S]. The more acidic terminal groups were expected to improve the enkephalin-like activity of the peptides. To study this question the opiate agonist potencies of the new compounds were

Primary structure and specificity of a serine proteinase inhibitor from paprika (Capsicum annuum) seeds

Several fractions demonstrating trypsin inhibitory activity were isolated from the seeds of the paprika plant (Capsicum annuum). One of the inhibitors, PSI-1.1, was purified to homogeneity and characterised. The mature form of PSI-1.1 has a molecular mass of 6053 Da and consists of 55 amino acids in a sequence showing over 80% identity with members of the inhibitors of potato-2 family. PSI-1.1 is a potent inhibitor of trypsin (Ki = 4.8 x 10(-10) M) and a somewhat weaker inhibitor of chymotrypsin (Ki = 4.7 x 10(-8) M) and pronase E (Ki = 5.9 x 10(-8) M). PSI-1.1 is resistant to heat up to 85 degrees C, to acidic conditions (down to pH 2.0) and to pepsin digestion, presumably due to its four disulfide bridges.

Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis

The gene of ecotin, an E. coli proteinase inhibitor, was cloned, and by site‐directed mutagenesis the active site residue of the protein, Met84, was mutated to Lys, Arg and Leu. The recombinant wild‐type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.

Is there correlation between analgesic potency and biodegradation of enkephalin analogs

Abstract Met-enkephalin and its Pro 5 analogs containing Gly or D amino acids at position 2 were subjected to digestion with aminopeptidase M, rat brain extracts and human sera. The enzyme resistance of these peptides was compared with their analgesic activity determined in tail flick test after central and intravenous administrations. Our data did not reveal an unambigous correlation between the analgesic potency and metabolic stability of the analogs. This suggest that analgesic activity of synthetic peptides should be due to factors other than enzyme resistance /e.g. receptor binding and transport properties/.