Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.