Role of reducing terminals in unfractionated and low-molecular-mass heparins in causing free radical generation and loss of structure and activity of trypsin.

The role of both the length of saccharide chain and reducing terminals in the heparin molecule in causing oxidative effects on proteins was investigated by employing unfractionated and low-molecular-mass heparins (LMMH), with both intact and reduced reducing terminals on bovine trypsin. The effects of heparin were found to be dependent on both the concentration and time of incubation. Heparins with intact reducing terminals caused significantly higher structural and functional alterations of trypsin compared with heparins with reduced reducing terminals. LMMH was slightly more effective than unfractionated heparin (UNFH) in reducing structural integrity and inhibiting the amidolytic activity of trypsin when used at the same mass, but not molar concentrations. Neither the length of saccharide chains nor the number of intact reducing terminals on the heparin molecule appeared to influence the characteristics of the initial binding of heparin to trypsin, but both these variables crucially affected linkages which in time mediate the inhibition of catalytic activity and the formation of free radicals, ultimately responsible for peptide bond cleavage in trypsin. The results suggest that both a critical number of saccharide units, preferentially lying on shorter chains, and intact reducing terminals in the heparin molecule are involved in setting up the binding which generates radicals and leads to loss of structure and function of the proteinase.     

Differential mechanisms for structural and functional alterations of trypsin by heparin, evidence for a specific, radical-generating mechanism at low heparin concentrations

The oxidative mechanism whereby heparin may interact with various proteins was investigated in detail in this work by addressing the role of doses of heparin on the nature and effects of its binding to bovine trypsin, taken as reference protein. Unfractionated heparin was used at concentrations ranging from 6 to 400 microg/ml with a fixed trypsin concentration (250 microg/ml). At concentrations of up to 60 microg/ml, equivalent to trypsin/heparin molar ratios of between 30 and 3, increasing inhibition of amidolytic activity and radical-dependent peptide bond cleavage of the enzyme was observed, with the appearance in the electrophoretic pattern of new bands of trypsin fragments to which heparin was demonstrated to be bound specifically. Structural modifications were also revealed by increases in fluorescence emission spectra. On the whole, however, the alterations induced by these heparin concentrations only involved a limited number of trypsin molecules. At concentrations from 120 to 400 microg/ml (equivalent trypsin/heparin molar ratios of 1.5-0.46), heparin binding to trypsin appeared to cause more profound and generalized alterations of enzyme structure and function, with dose-dependent quenching of fluorescence emission and almost complete loss of amidolytic activity, although evidence of radical production was lacking. Collectively, the results stress the crucial role of heparin dose on both the nature and effects of its binding to trypsin. The change in heparin effects which reflects distinct underlying molecular mechanisms occurs dramatically at a critical concentration threshold. While a specific, radical-generating mechanism operates at low concentrations, less specific ionic linkages, apparently independent of radical production, best explain the effects of high heparin concentrations.     

The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation

Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant.     

Investigation of the interaction of trypsin with heparin

The reversible inhibition of the enzymatic activity of trypsin by heparin was investigated. On the basis of an analysis of the Lineweaver-Burk and Dixon graphs, a noncompetitive nature of the inhibition of the BAPA amidase activity of trypsin by heparin was detected, and the values of Km and Ki were determined, equal to 3.1 . 10(-4) and 3.7-3.9 . 10(-7) M, respectively. A comparison of these values indicates a great affinity of heparin for the enzyme. It was shown that heparin inhibits the BAEE esterase activity of trypsin and at the same time has no inhibiting effect on acetyltrypsin. Considering that the acetylation of trypsin leads to selective blocking of the epsilon-amino groups, it was concluded that the epsilon-amino groups of the lysine residues of the trypsin molecule participate in the interaction with heparin.     

Effects of Ca2+ on catalytic activity and conformation of trypsin and alpha-chymotrypsin in aqueous ethanol

The effects of calcium ions on the conformation and catalytic activity of trypsin and alpha-chymotrypsin were studied in aqueous ethanol. The activity of alpha-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of alpha-chymotrypsin did not decrease in the presence of 1.2M CaCl2 and 0.6M CaCl2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.     

Some peculiarities of trypsin and heparin interaction]

The interaction of trypsin with an acid polysaccharide, heparin, at pH 4.2 and 8.0 is studied. Heparin is found to destabilize the enzyme under condition of both autolytic denaturation (pH 8.0) and thermoinactivation (pH 4.2). Data on trypsin inactivation kinetics suggest that the stage of forming molecular complexes with different contents of trypsin and heparin precedes the stage of the enzyme denaturation. Maximal trypsin inactivation rate takes place under equimolar enzyme:heparin ration.     

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.     

Modulatory effects on proteinase kinetics caused by association of both enzyme and substrate to heparin

Heparin is shown to produce modulatory effects on the amidolytic activity of trypsin, thrombin and plasmin with various synthetic peptide substrates. Simple Michaelis-Menten kinetics are observed in the absence of heparin. In its presence an enhancement effect is observed at low substrate concentrations, and an inhibitory effect is observed at high substrate concentrations. Other polyanions like dextran sulphate, phosvitin and inositol hexakisphosphate produces a similar effect. The modulatory effect of heparin is abolished when it binds cations. Co-binding of both substrate and enzyme to heparin seems to be a necessary requirement for the effect to occur. A model is proposed which can account semiquantitatively for the kinetics observed. It is suggested that the mechanism, which involves co-binding of substrate and enzyme in an competitive manner to a macromolecular structure, may be of primary importance as a regulatory mechanism in blood coagulation and fibrinolysis.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A.

Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.     

Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium

A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.     

Effect of the interphase surface charge on the structure and activity of trypsin in inverted micelles

The effect of the sign and value of the charge of the interphase surface on the catalytic activity of trypsin in systems of inverted mycelles was investigated. n-Butanol was used for the modification of the phase interface in dispersions of inverted mycelles based on anionic sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cationic cetyltrimethylammonium bromide (CTAB). A direct correlation between changes in the state of inverted mycelles and the structure of solubilized enzyme under the action of butanol was obtained. It was shown that the enzyme activity is determined by the quantity of butanol solubilized by the inverted mycelles.     

Ecotin modulates thrombin activity through exosite-2 interactions

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human alpha-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin-alpha-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC50=3.2 microM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC50=4.5 microM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-alpha-thrombin (EC50=3.5 microM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.     

Effect of the charge of the interface surface on the structure and activity of trypsin in reversed mycelles

The effect of the sign and value of the charge of the interface surface on the catalytic activity of trypsin in systems of reversed mycelles was investigated. n-Butanol was used for the modification of the phase interface in dispersions of reversed mycelles based on anionic sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cationic cetyltrimethylammonium bromide (CTAB). A direct correlation between changes in the state of reversed mycelles and the structure of solubilized enzyme under the action of butanol was obtained. It was shown that the enzyme activity is determined by the quantity of butanol solubilized by the reversed mycelles.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

Antigenicity of proteinases from Streptomyces griseus (pronase)

The five pronase fractions, A(1), A(2), B, C (trypsin-like), and D (elastolytic), obtained by ion-exchange chromatography, were found to be antigenically distinct. Antibodies to pronase inhibited the enzymic activity of each of the enzyme fractions. Pronase trypsin and bovine trypsin, although resembling each other in enzymic activity and in amino acid sequence around their active sites, did not cross-react antigenically with, nor was their enzymic activity inhibited by, the respective homologous antibodies. Inactivation of pronase trypsin by complexing with soya-bean inhibitor AA, was not associated with a decrease in capacity to precipitate with its antibody. It is assumed that the antigenic sites are located far enough from the catalytic site of the enzyme to allow it to precipitate immunologically even when the catalytic site was blocked.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.