Active-Site-Directed Inhibition of &α-Chymotrypsin by Deaminatively Produced Carbonium Ions: An Example of Suicide or Enzyme-Activated-Substrate Inhibition

Article abstract of DOI:10.1021/ja00404a043

N-Nitroso amides derived from phenylalanine and alanine were utilized as inhibitors of α-chymotrypsin.During the enzyme-catalyzed hydrolysis of these substrates, carbonium ions capable of alkylating nucleophilic groups are released in the active site.Nitroso lactams were also tested as substrates since they produce carbonium ions while still tethered to the enzyme at the acyl-enzyme stage.Kinetic studies indicated that at substrate/α-chymotrypsin ratios of 40:1 the acyclic substrates caused the following percent inhibition of α-chymotrypsin activity (substrate, percent inhibition): D-1a, 100; L-1a, <9; D-1c, 100; L-1c, 9.Nitroso lactams 2 and 3, in substrate/enzyme ratios of 54:1 and 20:1, respectively, caused 91 and 97percent inhibition of α-chymotrypsin activity.At low (<6:1) substrate/enzyme ratios, the inhibition of nitroso lactam 3 was partially reversible.The extents of inhibition were decreased by the competitive inhibitor N-acetyl-L-tryptophan, indicating that the inhibitor substrates were acting at the active site.Radioactive analogues of D- and L-1a and of 3(14C) provided evidence that the inhibition was irreversible, since ca. 1.0 mol of the benzyl group of D-1a and ca. 1.6 mol of the aryl moiety in the case of 3 remained bound to the inhibited enzyme after dialysis or Sephadex G-25 chromatography (no alkylation occurred with L-1a).The enzymatic hydrolyses of the L isomers of phenylalanine substrates (1a,b) were faster than those of the D enantiomers, whereas in the alanine series (1c) the rate ratio was reversed.A model based on "reverse" binding of the two aromatic groups of substrates D-1a and D-1c in the enzyme active site is proposed to explain the hydrolysis rate and the preferential inhibition of α-chymotrypsin by the D antipodes.