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PubMed2003

Spectroscopic studies on chromotropic mixed-ligand copper(II) complexes containing o-hydroxy benzoyl derivatives and dinitrogen bases.

Synthesis of chromotropic copper(II) mixed ligand complexes of o-hydroxy benzoyl derivatives (L1) and dinitrogen bases (L2), general formula Cun(L1)(L2)nXn; where n=1 or 2, L1=4,6-diacetyl-resorcinol (H2DACR), o-hydroxy benzaldhyde (HOHBZ) or o-hydroxy acetophenone (HOHAP), L2=N,N,N',N'-tetramethylethylenediamine (Me4en), 1,10-phenanthroline (phen) or bipyridine (bipy) and X=ClO4-, NO3- or Br- have been reported. Spectral, magnetic and molar conductance measurements as well as analytical data of these complexes show either mononuclear structure for OHBZ and OHAP or binuclear structure for DACR complexes. The IR stretching vibration frequencies of Cu-N and Cu-O are linearly correlated with the d-d absorption frequencies. The d-d absorption bands of Me4en-complexes in weak donor solvents suggest square-planar, distorted octahedral and distorted trigonal bipyramid geometries for the perchlorate, nitrate and bromide complexes, respectively. However, an octahedral structure is identified for the complexes in strong donor solvents. Perchlorate complexes show a remarkable color change from violet to green as the donor ability of the axial ligand increases, whereas bromide complexes are mainly affected by the Lewis acidity of the axial ligand. Specific and non-specific interactions of solvent molecules with the complexes are investigated using the unified solvation model. Calculations of the electronic transition probability (f) of the d-d band along with molecular orbital calculations of ligands have been carried out and correlated with the experimental data.

Chromogenic Compounds↗

PubMed1992

Effect of mastitis on plasminogen activator activity of milk somatic cells.

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

Animals↗

PubMed1995

Comparison of proteolytic activities in various lactobacilli.

A total of 169 Lactobacillus strains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9 Lactococcus lactis strains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between the Lactobacillus species and Lc. lactis. Lb. helveticus had by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases. Lb. delbrueckii subsp. bulgaricus possessed high hydrolysing activities towards substrates containing proline, alanyl-prolyl-p-nitroanilide and prolyl-p-nitroanilide. On the other hand, Lb. fermentum and Lb. brevis could be considered as weakly proteolytic species. A more detailed study with highly proteolytic Lactobacillus strains indicated that at least three different proteinases or endopeptidases were present. Compared with Lc. lactis, the Lactobacillus strains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.

Amino Acid Sequence↗

PubMed1992

Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates.

Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z-Phe-Arg-MCA. Two of these were optimally active at alkaline pH, the other at pH 4.0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz-Arg-MCA readily, whilst the other cleaved Bz-Arg-MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukaryotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc-Ala-Ala-pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz-Arg-MCA hydrolase, the aminopeptidases and the Boc-Ala-Ala-pNA serine peptidase are cytosolic.

Amino Acid Sequence↗

PubMed1998

A novel and simple colorimetric method for screening Giardia intestinalis and anti-giardial activity in vitro.

A new and simple colorimetric method has been developed for determining activity in vitro against Giardia intestinalis. The microtitre plate assay is based upon the nucleoside hydrolase activity released from G. intestinalis by lysis. Action of the nucleoside hydrolase on the substrate analogue, 4-nitrophenyl beta-D-ribofuranoside (NPR), gives rise to a coloured product which may be determined directly by the change in absorbance. A number of other such nucleoside analogues can be similarly used, but NPR is the preferred substrate, since it gives high enzymic activity at a relatively low substrate concentration. The IC50 values determined using this method for the known anti-giardials metronidazole, tinidazole and furazolidone were consistent with previously published values. The method is simple, does not involve radioisotopes or complex instrumentation, and thus provides a convenient method for screening potential anti-giardial agents.

Animals↗

PubMed2003

Cloning of and genetic variation in protease VCP1 from the nematophagous fungus Pochonia chlamydosporia.

The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root knot and cyst nematodes. It produces an alkaline serine protease, VCP1, during infection of nematode eggs. The gene encoding VCP1 was sequenced and the sequences of cDNAs from six isolates from different nematode hosts were compared. The gene encoding VCP1 was similar to PR1 from Metarhizium anisopliae with similar regulatory elements. Comparison of translated cDNA sequences revealed two amino acid polymorphisms at positions 65 and 99, indicating a difference between isolates from cyst and root nematodes. The positions and nature of the polymorphisms indicated that the two forms of VCP1 might have different properties and this was tested with five chromogenic polypeptide substrates. Enzyme assays revealed the two forms differed in their abilities to utilise Succ-Ala-Ala-Pro-Phe-pNa and Succ-Ala-Val-Pro-Phe-pNa, suggesting different amino acid affinities at the S3 binding region. This indicates host related genetic variation in VCP1 between isolates of P. chlamydosporia isolated from different nematode hosts, which might contribute to host preference. Such differences may be important in future exploitation of P. chlamydosporia as a nematode biocontrol agent.

Amino Acid Sequence↗

PubMed1996

Bifunctional labeling reagent for oligosaccharides to incorporate both chromophore and biotin groups.

We have developed a convenient and effective method for biotinylation of oligosaccharides at their reducing ends. A novel biotin hydrazide having a phenyl group produced the biotin adduct of N-acetyllactosamine (LacNAc) by simple incubation at 90 degrees C for 1 h. Although the biotin adduct was obtained as a mixture of several stereoisomers, one of the isomers, cyclic beta-glycoside, became predominant upon letting the reaction mixture stand in a weakly acidic state (pH 3.5). This conversion may be very advantageous for functional analysis of oligosaccharides because natural N-linked oligosaccharides exist in the cyclic beta form. The limit of detection of labeled LacNAc in reversed-phase chromatography was 330 fmol and showed good linearity in the range from 330 fmol to 261 pmol. When this procedure was applied to complex type and high mannose type N-linked oligosaccharides, the labeled oligosaccharides were easily detected and separated by reversed-phase, gel filtration, and anion exchange chromatographies. Furthermore, these labeled oligosaccharides were able to be immobilized onto the solid phase using avidin-biotin technology and were stable enough to allow the binding assay to be performed repeatedly and under the conditions for in situ exoglycosidase digestion. These results suggest that this derivatization technique might be useful for both separation and functional analysis of oligosaccharides.

Biosensing Techniques↗

PubMedDate not supplied

Chromogenic lactate-leukocyte esterase substrates.

The first successful reported use of lactate-based chromogenic, colorimetric substrates for a serine protease-based enzyme is described. A series of hydroxy-protected 5-phenyl-3-hydroxypyrrolyl L-lactate substrates of the general formula RO-Lac-OPP were prepared and formulated into reagents for the determination of leukocytes in dry phase formats.

Carboxylic Ester Hydrolases↗

PubMed1995

Photolysis of "purple" lipoxygenase: implications for the structure of the chromophore.

Treatment of soybean lipoxygenase isozyme 1 with its substrates, linoleic acid and oxygen, or product, 13(S)-hydroperoxy-9,11(Z,E)-octadecadienoic acid (13-HPOD), leads to the appearance of a purple color. Although the structure of the chromophore has not been determined, we present strong evidence that it is an Fe(3+)-OOR complex between the enzyme and 13-HPOD. Irradiation of frozen purple solutions of lipoxygenase causes the reversible production of a radical, shown by the effects of 2H and 17O enrichment on its EPR spectrum to be derived from 13-HPOD. The action spectrum of the photolysis reaction corresponds to the visible spectrum of the purple species, strongly implying that the purple chromophore contains 13-HPOD (or a product thereof) as part of its structure. Concomitant with the production of this radical there is a decrease in the intensity of an EPR signal corresponding to enzyme-bound Fe3+ and characteristic of the purple species. Taken together, these observations support the suggestion that the purple species is a complex between ferric lipoxygenase and 13-HPOD, likely the ferric peroxide.

Chromogenic Compounds↗

PubMed1993

Reaction of nerve growth factor gamma and 7S nerve growth factor complex with human and murine alpha 2-macroglobulin.

The kallikrein-like serine proteinase nerve growth factor gamma (NGF-gamma) reacted with the plasma proteinase inhibitor human alpha 2-macroglobulin (h alpha 2M). The h alpha 2M subunits were cleaved, the electrophoretic mobility of h alpha 2M in nondenaturing polyacrylamide gels was increased, and the intrinsic fluorescence of h alpha 2M was increased with a slight blue-shift. These changes are well-characterized components of the alpha 2M/proteinase reaction mechanism. In N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) hydrolysis experiments, the catalytic efficiency (kcat/KM) of the h alpha 2M-NGF-gamma complex was decreased by 98.5% compared with free NGF-gamma. This decrease is unique since other alpha 2M-proteinase complexes retain significant amidase activity. For comparison, we determined that the catalytic efficiency of alpha 2M-trypsin is decreased by 58% compared with free trypsin under equivalent conditions. The rate of NGF-gamma inhibition by h alpha 2M was (1.0 +/- 0.1) x 10(4) M-1 s-1 as determined by BAPNA hydrolysis. A similar value was determined by monitoring the change in intrinsic fluorescence. NGF-gamma, which was bound within the intact 7S NGF complex, also reacted with h alpha 2M, albeit at a very slow rate. This reaction may have depended exclusively on slow reversible dissociation of NGF-gamma from the 7S complex. NGF-gamma was rapidly inhibited by murine alpha 2M (m alpha 2M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Thrombin-fibrinogen interaction: pH dependence and effects of the slow-->fast transition.

A recently developed strategy capable of measuring the equilibrium dissociation constant for thrombin-fibrinogen interaction has been used to explore the pH dependence of the interaction and the effects of thrombin conformational transitions. The dependence of fibrinogen binding to thrombin in the pH range 6-10 is bell-shaped and remarkably similar to that obtained in the case of the small synthetic amide substrate tosyl-Gly-Pro-Arg-p-nitroanilide-AcOH. Since the synthetic substrate contains no groups that can ionize in the pH range 6-10, the bell-shaped curve must reflect ionization reactions of two groups of the enzyme with pK1 = 7.53 +/- 0.09 and pK2 = 8.80 +/- 0.09. These groups can be identified as the catalytic histidine, His57, and the amino terminus of the B chain, Ile16, respectively. Deprotonation of His57 in the acidic region is important for optimal binding, while protonation of Ile16 in the alkaline region is critical for the formation of a salt bridge with Asp194, which guarantees the conformational stability of the enzyme. The loss of binding free energy at low (< 7.0) and high (> 9.0) pH values is linked to protonation of His57 and deprotonation of Ile16, respectively. The first 51 residues of the A alpha chain of fibrinogen are known to be necessary and sufficient for optimal recognition by thrombin, but none of them contributes to the pH dependence of fibrinogen binding in the pH range examined. Hence, the two possible ionizable groups of the A alpha chain, i.e., the amino terminus Alal and His24, make no contacts with the thrombin surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗

PubMed1991

Mechanistic studies on thrombin catalysis.

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation.

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.

Amino Acid Sequence↗

PubMed2000

Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates.

Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophe nyl azo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role.

Amino Acid Sequence↗

PubMed1992

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.

Acyl Coenzyme A↗

PubMed2001

Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2.

Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites. Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release). This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds. k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate. We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation. The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2. These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences. This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins.

Acylation↗

PubMed2001

Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR.

A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned. The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme. The transcript has a signal peptide of 15 amino acid residues. The mature enzyme has 41% sequence identity with prostate specific antigen (PSA). Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies. The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222). Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond. These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.

Amides↗

PubMed2001

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Amino Acid Substitution↗