(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

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Abstract
(p-Amidinophenyl)methanesulfonyl fluoride (p-APMSF) was synthesized. It is a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor [F] Xa, human plasmin, human C1.hivin.r [activated r fragment of complement component 1] and human C1.hivin.s. The Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 .mu.M. p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF was compared with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with FXa, C1.hivin.r and C1.hivin.s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 .mu.M for thrombin but is a poor inhibitor of trypsin, FXa, C1.hivin.r, C1.hivin.s and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the DFP reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. The primary substrate binding sites of these enzymes, which share a high degree of structural homology, may significantly differ from each other in their ability to interact with low MW inhibitors and synthetic substrates.

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