Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate

Abstract

Serpins form a family of structurally related proteins, many of which function in plasma as inhibitors of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. To further characterize the mechanism by which serpins inhibit their target enzymes, we have studied the effect of temperature on the reaction of C1 inhibitor and the serine protease plasma kallikrein. At both 38 and 4 degrees C, C1 inhibitor (Mr 105,000) is cleaved by alpha-kallikrein (Mr 85,000 and 88,000) at position P1 (Arg444) of the reactive center, a reaction that leads to the formation of a covalent bimolecular enzyme-serpin complex (Mr 195,000) and cleaved but uncomplexed serpin (Mr 95,000). Between 38 and 4 degrees C, the product distribution is temperature-dependent, with more cleaved C1 inhibitor (Mr 95,000) formed at lower temperatures and correspondingly less Mr 195,000 complex. Studies employing intrinsic tryptophan fluorescence and 1H NMR spectroscopy show that this behavior is not caused by temperature-dependent conformational changes of kallikrein or C1 inhibitor. C1 inhibitor also behaves in this manner with the light chain of kallikrein and, to a lesser extent, with plasmin and C1s. These data are best explained by a branched reaction pathway, identical with the scheme describing the mechanism of action of suicide substrates. This scheme involves the formation of an enzyme-inhibitor intermediate, which can be stabilized into a covalent complex and/or dissociate into free enzyme and cleaved inhibitor, depending on the reaction conditions.

Interaction of subtilisins with serpins

“…Partitioning between the inhibitor and substrate pathways is seen for the interaction of serpins with some chymotrypsin family members (Patston et al, 1991;Gettins et al, 1992;Cooperman et al, 1993;Enghild et al, 1993;Schechter et al, 1993). However, it is not usually as extreme as shown here with the subtilisins unless specific mutations are made in the serpin that cause it to favor the substrate pathway (see Hood et al, 1994, for example).…”

“…Partitioning is most frequently due to cleavage predominating over inhibition at the PI site (Patston et al, 1991;Cooperman et al, 1993). Occasionally, cleavage outside the P I inhibitory site, but within the RSL, causes partitioning (Schechter et al, 1989;Enghild et al, 1993).…”

“…Some authors have concluded that this may represent the results of serpins acting as mechanism-based inactivators (Patston et al, 1991;Gettins et al, 1992;Lawrence et al, 1995). In this case, Met 358-Ser 359 cleavage would be an absolute requirement for inhibition and the inhibited proteinase would exist with its catalytic serine acylated by the a-carbonyl of Met 358 (Lawrence et al, 1995;Wright & Scarsdale, 1995).…”

“…The ratio of the inhibitor to the proteinase at the point of zero residual activity is defined as the partition ratio (Patston et al, 1991). Individual partition ratios are described by the intersection of the titration lines with the abscissa in Figure 1.…”

Caspase inhibitors: viral, cellular and chemical

“…It is thought that some CrmA molecules are cleaved and subsequently released from caspase 1, whereas other CrmA molecules are not cleaved but form an irreversibly stable complex. 13,33 …”