The sequence of alpha 1-antitrypsin is in keeping with its role as a tissue scavenger of leukocyte elastase. Two abnormal variants commonly present in Europeans cause a deficiency that predisposes them to a progressive loss of lung elasticity. The nature of the reactive centre helps explain why cigarette smoking greatly accelerates the onset and severity of this degenerative process to give the disease emphysema.
An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.
Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant protein's being an acute-phase reactant, the level of which increased several-fold after trauma.
Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A--sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P 1 Arg of ␣-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P 1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P 1 Arg becomes accessible. Furthermore, the deimination of P 1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P 1 His variant, due to a lowered dissociation rate constant for the antithrombinpentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P 1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P 1 Arg.The plasma serpin (1), antithrombin, is the major inhibitor of the serine proteinases of the coagulation network, especially thrombin and factor Xa (2). This inhibitory activity is stimulated by the complexing of antithrombin with the sulfated polysaccharide, heparin. In particular, the activity against factor Xa is mediated by a core pentasaccharide component of heparin. Antithrombin has an initial low affinity for heparin, but this immediately changes to a high affinity on initial binding, the change occurring concomitantly with the activation of inhibitory function (3).The interaction of the heparin pentasaccharide with antithrombin and the associated mechanism of conformational activation of inhibition has recently been revealed in the crystal structure of a dimer of antithrombin complexed with the pentasaccharide (4). Linkage between the two antithrombin molecules in the dimer directly involves the reactive site loop of the inhibitory component, with a consequent constraint in the movement at its reactive site. Hence, while showing the commencement of the movement of the reactive site loop that results in activation, the crystal structure does not show the change that occurs at the reactive center itself.There is, however, a good model of the likely unconstrained active conformation of the reactive site loop of antithrombin. This is provided by the structure of the closely related serpin, ␣ 1 -antitrypsin (5), in which the loop is fixed in the optimal canonical inhibitory conformation present in all other families of serine proteinase inhibitors. The transition of antithrombin to this conformation would require a shift of the side chain of the P 1 arginine from an i...
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