Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.     

The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.     

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD⁺) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP⁺, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD⁺ reaction of reconstituted enzyme. NAD⁺ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H⁺ translocation that was comparable to the decrease in the rate of NADPH → AcPyAD⁺ transhydrogenation. It is concluded that tetranitromethane modification controls the H⁺ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H⁺-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.     

Effect of thrombomodulin on specific thrombin functions: fibrinogen coagulation and thrombocyte aggregation

The effect of thrombomodulin (TM), prepared from rabbit lungs, on fibrinogen clotting and platelets aggregation by alpha-thrombin has been investigated. It has been established that TM caused a dose-dependent decrease in fibrinogen-clotting activity of thrombin (Ki = 14.7 +/- 1.24 nM). TM was shown to reduce thrombin-induced platelet aggregation but not to alter ADP-induced one. It was found that the kinetic parameters for hydrolysis of synthetic substrates by alpha-thrombin were not altered by TM.     

Macromolecular specificity determinants on thrombin for fibrinogen and thrombomodulin

The endothelial cell surface membrane protein thrombomodulin binds thrombin with high affinity and acts as both a cofactor for protein C activation and an inhibitor of fibrinogen hydrolysis. We have previously shown that bovine thrombomodulin is a competitive inhibitor of fibrinogen binding to thrombin but has no effect on thrombin activity toward tripeptide substrates or antithrombin III. Hence, thrombomodulin and fibrinogen may share macromolecular specificity sites on thrombin which are distinct from the active site. In this investigation, we have studied the interaction of thrombin-thrombomodulin with fibrinogen and various thrombin derivatives. We show that fibrinogen is a competitive inhibitor of thrombomodulin binding to thrombin, with a Kis = 10 microM. Thrombin derivatives (bovine (pyridoxal phosphate)4-thrombin and human thrombin Quick I), which bind fibrinogen with much reduced affinity, are shown to also interact with thrombomodulin with greatly reduced affinity. These results are consistent with the hypothesis that thrombomodulin and fibrinogen share macromolecular specificity sites on thrombin.     

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.     

Three copies of the beta subunit must be modified to achieve complete inactivation of the bovine mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyladenosine

The modification of both beta-Tyr-368 and beta-His-427 can be correlated with the loss of activity observed when the bovine mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoyl[3H]adenosine ([3H]FSBA). At pH 8.0, where the rate of inactivation is fast, beta-Tyr-368 is modified predominantly, while at pH 6.0, where the rate of inactivation is slow, beta-His-427 is modified predominantly. At pH 7.0, the 2 residues are modified with about equal efficiency. When the F1-ATPase was inactivated by 80% at pH 6.5, 7.0, and 7.5, the sum of radioactivity incorporated into beta-Tyr-368 and beta-His-427 was 1.99, 1.87, and 1.82 mol of label incorporated per mol of enzyme, respectively. Examination of the rate of inactivation of the enzyme by FSBA as a function of pH revealed two pKa values, one of about 7.6 associated with the modification of beta-Tyr-368 and the other of about 5.8 associated with the modification of beta-His-427. The inactivation of the F1-ATPase by FSBA exhibited an initial fast rate followed by a slower rate in triethanolamine-HCl, pH 7.0. In contrast, only a single rate, equivalent to the fast phase of inactivation in the absence of phosphate, was observed in 0.2 M phosphate, pH 7.0. The dependence of this stimulation on phosphate concentration is sigmoidal with half-maximal stimulation occurring at approximately 160 mM. The ratio of 3H incorporated into beta-Tyr-368 to that incorporated into beta-His-427 was approximately the same during the fast and slow phases of inactivation in triethanolamine-HCl, pH 7.0. Approximately the same ratio was observed when the enzyme was modified during the single phase of inactivation exhibited in the presence of 0.2 M phosphate, pH 7.0. The sum of the 3H incorporated into beta-Tyr-368 and beta-His-427 during inactivation of the F1-ATPase from bovine heart mitochondria by [3H]FSBA in the presence and absence of phosphate was linear and extrapolated to a value of about 2.6 residues modified on complete inactivation of the enzyme. From these data, it is concluded that FSBA binds to a single binding site on the beta subunits of the enzyme where it reacts with either beta-Tyr-368 or beta-His-427 in mutually exclusive reactions. All three beta subunits must be modified in this manner for complete inactivation to be observed.     

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

Phenylglyoxal modification of cardiac myosin S-1. Evidence for essential arginine residues at the active site.

The role of arginine residues in the catalytic activity of cardiac myosin subfragment-1 (S-1) was investigated by selective modification with phenylglyoxal. Incorporation of about 2.8 mol of phenylglyoxal/mol of S-1 decreased Ca2+-ATPase activity about 50%. Gelation of the protein occurred at about 70% inactivation; however, extrapolation to complete inactivation indicated that loss of activity correlated with modification of about 4 arginyls/mol. Partial inactivation of S-1 with phenylglyoxal also decreased MgADP binding markedly. When S-1 was modified in the presence of 5 mM MgADP, only 2 arginyls/mol were blocked and there was almost complete protection against loss of Ca2+-ATPase activity and ability to bind MgADP. Similar protection against inactivation by phenylglyoxal was obtained with MgATP or sodium pyrophosphate, but not with MgAMP or magnesium adenosine. These results suggest that 2 arginyls/myosin head are important for enzymatic activity, possibly serving as attachment points between enzyme and substrate. These essential arginyls were localized to a 17,000-dalton cyanogen bromide peptide from the heavy chain fragment of S-1.     

Characterization of the catalytic defect in the dysthrombin, Thrombin Quick

The dysthrombin, Thrombin Quick, is chromatographically separable into two components designated Thrombin Quick I and Thrombin Quick II. Thrombin Quick II lacks observable catalytic activity toward thrombin substrates. The steady-state kinetics of hydrolysis of benzoylarginine ethyl ester and Tos-Gly-Pro-Arg-p-nitroanilide by Thrombin Quick I are equivalent to those of thrombin. These results, in addition to binding studies with the active site titrant N2-(5-dimethylaminonaphthalene-1-sulfonyl)arginine N-(3-ethyl-1,5-pentanediyl)amide, indicate that binding interactions at the catalytic site of Thrombin Quick I are unaltered. Thrombin Quick I is inhibited by anti-thrombin III at the same rate as thrombin. Steady-state kinetic parameters for the release of fibrinopeptide A indicate defects in both kcat and Km for Thrombin Quick I with kcat/Km equal to 0.012 of the value for thrombin, corresponding to the relative fibrinogen clotting activity of 0.013. The results are interpreted as indicating a defect in Thrombin Quick I at a binding site, external to the catalytic site, which is essential for determining specificity toward fibrinogen. The defect in kcat may result secondarily from small perturbations in the steric relationship of the catalytic triad residues. The rate of hydrolysis by Thrombin Quick I of the protein substrates bovine prothrombin and bovine protein C (in the absence of cofactors) is about one-third of that observed for thrombin, indicating that hydrolysis of these substrates by thrombin involves different specificity determinants than does the hydrolysis of fibrinogen.     

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.     

Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane

Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.     

Mutations in autolytic loop-2 and at Asp554 of human prothrombin that enhance protein C activation by meizothrombin

Thrombin acts on many protein substrates during the hemostatic process. Its specificity for these substrates is modulated through interactions at regions remote from the active site of the thrombin molecule, designated exosites. Exosite interactions can be with the substrate, cofactors such as thrombomodulin, or fragments from prothrombin. The relative activity of alpha-thrombin for fibrinogen is 10 times greater than that for protein C. However, the relative activity of meizothrombin for protein C is 14 times greater than that for fibrinogen. Modulation of thrombin specificity is linked to its Na(+)-binding site and residues in autolytic loop-2 that interact with the Na(+)-binding site. Recombinant prothrombins that yield recombinant meizothrombin (rMT) and rMT des-fragment 1 (rMT(desF1)) enable comparisons of the effects of mutations at the Na(+)-binding residue (Asp(554)) and deletion of loop-2 (Glu(466)-Thr(469)) on the relative activity of meizothrombin for several substrates. Hydrolysis of t-butoxycarbonyl-VPR-p-nitroanilide by alpha-thrombin, recombinant alpha-thrombin, or rMT(desF1) was almost identical, but that by rMT was only 40% of that by alpha-thrombin. Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by alpha-thrombin, as already known. Strikingly, however, although meizothrombins modified by substitution of Asp(554) with either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of alpha-thrombin, the activity of these meizothrombins for protein C was increased to >10 times that of alpha-thrombin. It is proposed that interactions within thrombin that involve autolytic loop-2 and the Na(+)-binding site primarily enhance thrombin action on fibrinogen, but impair thrombin action on protein C.     

Functional consequences of tryptophan modification in human fibrinogen.

When human fibrinogen was modified with H2O2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.     

Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.     

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.