Aminopeptidases 323
2.2.2. Specificity
The enzyme cleaves N-terminal residues from all peptides having a
free a-amino or o~-imino group. However, in peptides containing an X---
Pro sequence, where X is a bulky hydrophobic residue (Leu, Tyr, Trp, Met
sulfone), or in the case of an N-blocked amino acid, cleavage does not
occur. It is for this reason that prolidase (Section 2.3.) is used in con-
junction with aminopeptidase M to produce total hydrolysis of peptides.
2.2.3. Molecular Mass
The enzyme has a mol mass of about 280,000 Da based on gel filtration
and is composed of l0 subunits (28,000 + 3000 Da) of two different
types (27). The enzyme molecule contains five disulfide bridges, each
of which connects two subunits (27). The gene has been cloned, the
amino acid sequence determined, and mol mass confirmed (27,28).
2.2.4. pH Optimum
At substrate concentrations used for sequence work, the pH opti-
mum is between 7.0 and 7.5, but at higher substrate concentrations,
approaches 9.0 (15).
2.2.5. Assay
The assay is based on hydrolysis ofp-nitroanilides of amino acids,
especially alanine or leucine. The enzyme is added to 0.06M phos:
phate buffer, pH 7.0, containing 1.66 mM L-leucine p-nitroanilide or
L-alanine-p-nitroanilide to give a final vol of 2.0 mL. The increase in
absorbance at 405 nm is recorded at 37°C. One enzyme unit is defined
as the amount of enzyme that produces 1 ~nol p-nitroanilide/min at
37°C (16). The enzyme is normally supplied with a specific activity of
4 U/mg protein (16). Alternatively, an assay based on the hydrolysis
of L-leucinamide at pH 8.5 and 25°C is used. Using this assay, the
enzyme is usually supplied with a specific activity of 25 U/mg protein.
The hydrolysis of phenylalanyl-3-thia-phenylalanine at pH 8.2 and
25°C has also been used as the basis of an assay that distinguishes
between leucine aminopeptidase and aminopeptidase M (29).
2.2.6. Stability
The lyophilized enzyme is stable for several years at -20°C. A
working solution can be prepared by dissolving about 0.25 mg of
protein in 1 mL of deionized water to give a solution of approx 6 U of
activity/mL. This solution can be aliquoted and stored frozen for sev-