The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Inhibition of prothrombinase complex by plasma proteinase inhibitors

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.     

Reaction of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase

The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M-1 X min-1 (dithiothreitol-sensitive reaction) and 52 M-1 X min-1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phosphoenolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1,N6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1,N6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.     

Reactivity and pH dependence of thiol conjugation to N-ethylmaleimide: detection of a conformational change in chalcone isomerase

The reactivity of simple alkyl thiolates with N-ethylmaleimide (NEM) follows the Br?nsted equation, log kS- = log G + beta pK, with G = 790 M-1 min-1 and beta = 0.43. The rate constant for the reaction of the thiolate of 2-mercaptoethanol with NEM is 10(7) M-1 min-1, whereas the rate constant for the reaction of the protonated thiol is less than 0.0002 M-1 min-1. The intrinsic reactivity of the protonated thiol (SH) is over (5 X 10(10]-fold less than the thiolate (S-) and makes a negligible contribution to the reactivity of thiols toward NEM. The rate of NEM modification of chalcone isomerase was conveniently measured by following the concomitant loss in enzymatic activity. The pseudo-first-order rate constants for inactivation show a linear dependence on the concentration of NEM up to 200 mM and yield no evidence for noncovalent binding of NEM to the enzyme. Evidence is presented demonstrating that the modification of chalcone isomerase by NEM is limited to a single cysteine residue over a wide range of pH. Kinetic protection against inactivation and modification by NEM is provided by competitive inhibitors and supports the assignment of this cysteine residue to be at or near the active site of chalcone isomerase. The pH dependence of inactivation of the enzyme by NEM indicates a pK of 9.2 for the cysteine residue in chalcone isomerase. At high pH, the enzymatic thiolate is only (3 X 10(-5))-fold as reactive as a low molecular weight alkyl thiolate of the same pK, suggesting a large steric inhibition of reaction on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible inhibition of bovine lung angiotensin I-converting enzyme with p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and chlorambucyl L-proline and with evidence that an active site carboxyl group is labeled

Bovine lung angiotensin I-converting enzyme is rapidly and irreversibly inactivated by p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and by the chlorambucil derivative of L-proline (chlorambucyl-proline). Chlorambucil is a nitrogen mustard alkylating agent that is used as an antineoplastic drug. At any one concentration, the inactivation is pseudo-first order with time. Inhibition by both substances is active site directed as suggested by the formation of a reversible enzyme-inhibitor complex prior to the alkylation reaction and by the fact that L-Phe-L-Pro, a reversible inhibitor which is competitive with substrate, is also competitive with both irreversible inhibitors in protecting the enzyme against inactivation. The second order rate constant for inactivation increases in the pH range 5-8 and reaches a value of 3.5 X 10(3) M-1 . min-1 for chlorambucil and 4.8 X 10(2) M-1 . min-1 for chlorambucyl-proline. Chlorambucyl [U-14C]L-proline reacts 1:1 with the converting enzyme and the uptake of radioactivity paralleled the loss of enzyme activity with and without protection by Phe-Pro. Once bound, the radioactive chlorambucyl proline was released (as the dihydroxy derivative) by hydroxide ion with a second order rate constant of 2.2 M-1 . min-1 at 25 degrees C. The radioactive label is also removed by hydroxylamine at pH 10. The lability of the irreversibly bound inhibitor in alkali and in hydroxylamine indicates that an ester bond is formed by the alkylation of an aspartic acid or glutamic acid side chain.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.     

The incorporation of a fluorescent probe into the active sites of one- and two-chain tissue-type plasminogen activator

Dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (DEGR-CK) was shown to inactivate both one- and two-chain human, recombinant tissue-type plasminogen activator (t-PA). The interaction of DEGR-CK with both forms of t-PA was accompanied by an identical increase in the fluorescence intensity and a blue shift in the wavelength of maximum emission, which suggests that the environment of the incorporated DEGR is similar in both one- and two-chain t-PA. The kinetics of the interaction of t-PA with DEGR-CK could be followed by both loss of activity and increase in fluorescence. The second order rate constants (k2/Ki) obtained with these two methods agreed quite well. With two-chain t-PA the values were 42 X 10(4) M-1 min-1 and 46 X 10(4) M-1 min-1 by the activity loss and fluorescence methods, respectively. With one-chain t-PA the results were 2.5 X 10(4) M-1 min-1 and 3.1 X 10(4) M-1 min-1. The rate at which one-chain t-PA is inactivated by DEGR-CK is 15 times lower than the rate with two-chain t-PA. The results demonstrated, however, that the cleavage of the one-chain protein to the two-chain form is not required for reactivity with DEGR-CK. This fluorescently labeled t-PA should be useful in probing the interactions of one- and two-chain t-PA with other proteins.     

tert.-Butyl aminocarbonate (tert.-butyloxycarbonyloxyamine)--a new acylating reagent for amines

tert.-Butyl amino carbonate (1, tert.-butyloxycarbonyloxyamine) was prepared by reaction of hydroxylamine with di-tert.-butyl dicarbonate (2). 1 was used to acylate different amino acids or amines in water or in aqueous dioxane. 1 was also prepared in situ and used to acylate amino acids directly. 1 reacted 1.5-2.5 times faster than 2 with all amines studied either in water or 50% (v/v) dioxane. Remarkably, 1 retained its ability to acylate amines even in acid solution; the rate of acylation of L-Phe at pH 6.5 (15 M-1 X min-1) was about 20% of the rate at pH 10 (72 M-1 X min-1). 2 was not an acylating reagent below pH 7.0 and as expected, the rate at pH 8.3 (4 M-1 X min-1) was about 10% of that at pH 9.3 (39 M-1 X min-1). Pure BOC-amino acids were obtained in high yield and could be quantitatively deprotected by standard procedures.     

The carboxylesterases of Trypanosoma cruzi epimastigotes

Carboxylesterase activity in Trypanosoma cruzi was found mainly in the microsomal (40%) and the cytosolic fraction (26%). The Vmax for p-nitrophenyl acetate was 28.50 and 17.60 nmol per min and mg of protein for the microsomal and the cytosolic fractions, respectively. The Km was 0.78 mM for the microsomal activity and 0.55 mM for the cytosolic activity. The inhibition rate constant with N-ethylmaleimide were 38.10 M-1 min-1 and 2.56 M-1 min-1 for the cytosolic and the microsomal enzymes, respectively. The rate constants with Paraoxon were 8,360 M-1 X min-1 and 32,600 M-1 X min-1. Polyacrylamide gel electrophoresis under nondenaturing conditions showed three bands of microsomal activity with M.W. of 63, 136 and 153 kDA. Similarly, three bands of cytosolic activity with M.W. of 126, 294 and 479 kDA were identified.     

The anticoagulant properties of mast cell product, chondroitin sulphate E

The anticoagulant potency in vitro of chondroitin sulphate E has been found to be similar to that of the heparinoids. In purified systems chondroitin sulphate E was shown to be principally an activator of heparin cofactor II. Maximum acceleration of heparin cofactor II:thrombin interaction was 185-fold (9.3 X 10(7) M-1 min-1), antithrombin III:thrombin interaction was 11-fold (4.16 X 10(6) M-1 min-1) and antithrombin III:factor Xa was 146-fold (3.86 X 10(6) M-1 min-1). Chondroitin sulphate E was observed to prolong the thrombin clotting time of fibrinogen in the absence of antithrombin III and heparin cofactor II. The effect appeared to be related to interference in thrombin:fibrinogen interaction rather than in fibrin monomer polymerization.     

The antiheparin effect of a heparinoid, pentosan polysulphate. Investigation of a mechanism.

A pentosan polysulphate [a fully sulphated (1-4)-beta-D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid] has been shown to inhibit the anticoagulant activity of high-affinity heparin as observed in plasma and when using purified enzyme and inhibitor. The activity was shown to be concentration-dependent with an apparent Ki of approx. 2 microM. The antiheparin property was not shown by a number of other anionic carbohydrates when tested. The rate of thrombin inhibition at 0.33 microM-heparin was reduced from 7.1 X 10(8) M-1 X min-1 in the absence of pentosan polysulphate to 2.3 X 10(8) M-1 X min-1 at 2 microM-pentosan polysulphate and to 0.3 X 10(8)M-1 X min-1 at 20 microM. Using the random bireactant model of heparin action [Griffiths (1982) J. Biol. Chem. 257, 13899-13902] it was observed that the pentosan polysulphate had no effect on the Km for antithrombin III (150 nM) but increased the Km for thrombin from 25 nM to 450 nM. A reduction in the inhibition rate by 17.3-fold predicted by substitution of these values into the general two-substrate reaction-rate equation was confirmed experimentally.     

Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.     

The relative molecular mass dependence of the anti-factor Xa properties of heparin.

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.     

The binding of zinc to angiotensin-converting enzyme

The equilibrium constant for the dissociation of zinc ion from angiotensin-converting enzyme (ACE) was measured using zinc ion buffers of zinc chloride and nitrilotriacetic acid (NTA). The dissociation constant is 6.4 X 10(-10) M. The fraction of active enzyme at equilibrium is independent of the presence of substrate which indicates that hippuryl-histidylleucine binds equally well to the holoenzyme and apoenzyme. The rate constant for the dissociation of zinc from ACE was measured as 0.68 min-1 for the free enzyme; the rate constant for the enzyme substrate complex was roughly 0.18 min-1. The association of zinc ion and ACE is very fast; the rate constant is 1.06 X 10(9) M-1 min-1. Ethylenediaminetetraacetic acid (EDTA) and NTA rapidly remove zinc from ACE with rate constants of 1.27 X 10(3) and 2.2 X 10(3) M-1 min-1. The equilibrium constant for the reaction of NTA with ACE was measured as 4.6 X 10(-2) and was calculated for EDTA as 3.8 X 10(3).     

Cooperative behavior in the thiol oxidation of rabbit muscle glycogen phosphorylase in cysteamine/cystamine redox buffers

Glycogen phosphorylase a and b are irreversibly inactivated by oxidation with the disulfide cystamine. The mechanism is complex and involves oxidation of at least two classes of sulfhydryl groups. The oxidation of one or more of the first class of 4 +/- 1 sulfhydryl groups is reversible, but the equilibrium constant for the oxidation is so unfavorable (1 X 10(-4)) that the micromolar concentrations of cysteamine released stoichiometrically with enzyme oxidation are sufficient to prevent complete oxidation even in the presence of 100 mM cystamine. The rapid phase of inactivation of phosphorylase b, which is first order in cystamine (k = 2.9 +/- 0.3 M-1 min-1), is followed by the oxidation of 5 +/- 1 groups in an irreversible process that is second order in cystamine concentration (k = 3.9 +/- M-2 min-1). Similar behavior is observed for phosphorylase a, although the behavior is complicated by association/dissociation equilibrium. The second-order dependence of the rate of irreversible inactivation on cystamine concentration is interpreted in terms of a "cooperative" model in which a rapidly reversible thermodynamically unfavorable equilibrium oxidation of one or more sulfhydryl groups must precede the irreversible oxidation of one or more additional sulfhydryl groups. The thiol/disulfide oxidation equilibrium constant for the initial reversible reaction is estimated to be at least 10(4) less favorable than that for the reversible oxidation of phosphofructokinase.     

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

Reactivity of the phosphopyridoxal groups of cystathionase.

Aminooxyacetate and alpha-amino-gamma-aminooxybutyrate (canaline) react specifically with the P-pyridoxal groups of cystathionase to produce characteristic changes in the absorption and fluorescence properties of the bound cofactor. The increase in fluorescence at 450 nm was used to monitor the reaction. Aminooxyacetate attacks the Schiff base linkage of the enzyme several times faster (k1 = 3700 M-1 min-1 and k2 = 1000 M-1 min-1) than it attacks the aldehydic carbon of free P-pyridoxal (k = 290 M-1 min-1). Similar results were obtained with canaline. The kinetic studies indicate that a Schiff base linkage in the enzyme cystathionase should offer direct kinetic advantage during the reaction between the substrate and the cofactor. It is also shown that the inhibitor L-alpha-gamma-aminobutyrate reacts with bound P-pyridoxal to form free P-pyridoxamine. The rate of formation of P-pyridoxamine parallels the rate of enzyme inactivation.     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.