Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate.
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.
Patston
P A
PA
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232.
Gettins
P
P
Beechem
J
J
Schapira
M
M
D003174:Complement C1 Inactivator Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D015843:Serpins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D013696:Temperature
D003174:Complement C1 Inactivator Proteins; D010446:Peptide Fragments; D015843:Serpins; D007610:Kallikreins