INACTIVATION OF PAPAIN BY ANTITHROMBIN DUE TO AUTOLYTIC DIGESTION - AMODEL OF SERPIN INACTIVATION OF CYSTEINE PROTEINASES

Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable s erpin-cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation o f the cysteine proteinases, papain and cathepsin L, by the serpin anti thrombin. The two proteinases were inactivated with second-order rate constants of (1.6+/-0.1) x 10(3) and (8.6+/-0.4) x 10(2) M-1.s(-1) res pectively. An antithrombin to papain inactivation stoichiometry of sim ilar to 3 indicated extensive cleavage of the inhibitor concurrent wit h enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal se quence analyses showed cleavage predominantly at the P-2-P-1 bond, but also at the P-2'-P-3' bond of antithrombin. The papain band in SDS/ P AGE progressively disappeared on reaction of the enzyme with increasin g amounts of antithrombin, but no band representing a stable antithrom bin-papain complex appeared. SDS/PAGE with I-125-labelled papain showe d that the disappearance of papain was caused by cleavage of the enzym e into small fragments. These results suggest a mechanism in which pap ain attacks a peptide bond in the reactive-bond loop of antithrombin a djacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, t o form an inactive acyl-intermediate complex, although with the substr ate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and result s in liberation of fragmented, inactive enzyme. This model may have re levance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.