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Effects of pH on the Michaelis constant and maximal velocity of hydrolysis of l‐alanine‐4‐nitroanilide by aminopeptidase N have been studied in the range between pH 6.5 and pH 9.5. V does not vary with pH in this range, therefore any ionizable group shown to be involved in the action of aminopeptidase N will likely be affecting binding and not the catalytic step. The pKmvs pH profile suggests that two ionizable groups (pKa= 7.2 and pKa= 8.2) affect binding of this substrate. The pKivs pH profile observed with the competitive inhibitor hexylamine does not show any inflection point in the alkaline region (pH > 7.2), which suggests that the pKa= 8.2 at 37°C corresponds to the pKa of the substrate ammonium group. In the neutral pH range the inflection points occur at pH 7.2 both in plots of pKmvs pH and pKivs pH. Thus the corresponding ionizable group should belong to the enzyme. Since the strategic–SH group of aminopeptidase N is protected from the action of N‐ethylmaleimide by hexylamine and other competitive inhibitors, we propose that the value of pKa= 7.2 corresponds to the ionization of this group. Aminopeptidase N is competitively inhibited by l‐amino acids having hydrophobic side‐chains such as leucine, isoleucine, phenylalanine, tyrosine and tryptophan. Blocking of the α‐amino group prevents binding, and blocking of the α‐carboxylic group reduces the inhibitory capacity of the amino acid. Aliphatic amines are also competitive inhibitors; the longer the aliphatic chain, the smaller the Ki, which varies by a factor 20 from butylamine to octylamine. Even though aminopeptidase N is not a metalloenzyme, divalent cations competitively inhibit the hydrolysis of l‐alanine‐4‐nitroanilide. The efficiency of binding is in the order: Cd2+ > Zn2+ > Cu2+ > Co2+ > Mn2+ > Mg2+ > Ca2+ with Cd2+ ions having a 103 higher binding constant than Ca2+ ions. The Ki values and initial rates of hydrolysis of 15 peptides by aminopeptidase N are reported. The peptides tested included: alanyl‐X, X‐alanine, di, tri and penta‐alanine, and dipeptides devoid of alanine, where X represents any one of several amino acids. The C‐terminal amino acid of alanyl‐X dipeptides contributes mainly to the binding constant, which depends on the hydrophobicity of their side‐chain. This affinity varies by a factor of 28 from glycine to phenylalanine whereas V varies only by a factor of 2. Effects of changes in the NH2‐terminal amino acid in X‐alanine dipeptides and in dipeptides devoid of alanine on the rate of hydrolysis and Ki, was also studied. V values for the enzyme of dipeptides devoid of alanine decreased with Ki of the peptides. For di, tri, and pentaalanine, the increase of the peptide size increases the affinity but not the rate of hydrolysis.

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