Aminopeptidase N from Escherichia coli: Ionizable Active‐Center Groups and Substrate Specificity

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 p K m vs pH profile suggests that two ionizable groups (p K a = 7.2 and p K a = 8.2) affect binding of this substrate. The p K i vs 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 p K a = 8.2 at 37°C corresponds to the p K a of the substrate ammonium group. In the neutral pH range the inflection points occur at pH 7.2 both in plots of p K m vs pH and p K i vs 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 p K a = 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 K i , 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: Cd 2+ > Zn 2+ > Cu 2+ > Co 2+ > Mn 2+ > Mg 2+ > Ca 2+ with Cd 2+ ions having a 10 3 higher binding constant than Ca 2+ ions. The K i 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 NH 2 ‐terminal amino acid in X‐alanine dipeptides and in dipeptides devoid of alanine on the rate of hydrolysis and K i , was also studied. V values for the enzyme of dipeptides devoid of alanine decreased with K i of the peptides. For di, tri, and pentaalanine, the increase of the peptide size increases the affinity but not the rate of hydrolysis.