Hydrophobic binding sites of human liver alanine aminopeptidase

Fatty acids, alkyl amines, and amides of α-amino fatty acids inhibit human liver alanine aminopeptidase apparently by binding to residue binding site 1 of the active center, i.e., the N-terminal binding site. The pK_i values of the acids, amines, and amides increase until the overall chain length reaches eight carbons. The pK_i values are the same for members of the series with chain lengths longer than eight carbon atoms. Assuming an extended structure of the inhibitors, this site will accommodate amino acid side chains of not longer than 11.7 Å from the α-carbon to the end of the chain. Long chain amino acids inhibit by binding apparently at residue site 3. The pK_i values of dl-α-amino acids from α-aminobutyric acid to α-aminodecanoic acid increase with the addition of each methylene unit. Thus, site 3 will accommodate amino acid side chains which are at least 13.0 Å from the α-carbon to the end of the chain. Methanol and other organic solvents reversibly inhibit the binding of substrates at pH 6.9 without affecting the maximum rate of catalysis. At lower pH values, the maximum rate of catalysis is lowered. Sodium chloride also inhibits substrate hydrolysis at pH 6.9 but does not affect the maximum rate of catalysis. The pK_i values of fatty acids, alkyl amines, and amino acids are strongly decreased by methanol and slightly increased by sodium chloride. These data indicate that a major portion of the interactions of the enzyme with fatty acids, amines, and amino acids is of a hydrophobic nature.