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Kinetic analysis of the heparin-enhanced plasmin--antithrombin III reaction. Apparent catalytic role of heparin.

ABSTRACT: Inactivation of plasmin by a 3-4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5mum and 1mum respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin-antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin-plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain ;optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin-antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2mug/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.

SUBMITTER: Machovich R

PROVIDER: S-EPMC1163406 | biostudies-other | 1981 Dec

REPOSITORIES: biostudies-other

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Project description:The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

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Kinetic analysis of the heparin-enhanced plasmin--antithrombin III reaction. Apparent catalytic role of heparin.

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