Aminopeptidases 321
- Enzyme Data
2.1. Pyroglutamate Aminopeptidase (Calf Liver)
2.1.1. Alternative Names
These are L-pyroglutamyl peptide hydrolase, 5-oxoprolyl-peptidase,
pyrrolidonyl peptidase, pyroglutamate aminopeptidase, pyroglutamyl
peptidase, and pyroglutamase. Note: This enzyme is now classified as
EC 3.4.19.3. In earlier literature, the enzyme was classified as EC 3.4.11.8.
2.1.2. Specificity
The enzyme cleaves N-terminal pyroglutamyl residues from pep-
tides and proteins, but not if the following residue is proline. The
enzyme has highest specificity when the pyroglutamate residue is
linked to alanine (13).
2.1.3. Molecular Mass
A mol mass of 79,000-80,000 Da has been reported for the enzyme,
based on gel filtration studies (21).
2.1.4. pH Optimum
The enzyme is active at pH 7-9, but is normally used at pH 8.0 (22).
2.1.5. Assay
The assay is based on the hydrolysis of L-pyroglutamic acid 13-naph-
thylamide. The enzyme buffer used is prepared as follows: 1 L of 0.1M
Na2HPO 4 is adjusted to pH 8 with 0.1M NaH2PO 4 to give 1605 mL,
and this solution is made 5% (v/v) in glycerol. A deblocking buffer is
prepared by making 112 mL of the enzyme buffer, 5 mM in DTT, and
10 mM in Na 2 EDTA, adjusting the solution to pH 8 and purging with
N 2. Enzyme solution (0.4 mL) and 0.5 mL of deblocking buffer are
combined, purged with argon, and sealed with a cap. The mixture is
incubated for 3 min at 37°C, the substrate added (1.78 mmol
pyroglutamyl-~-naphthylamide in 0.1 mL of methanol), and the mix-
ture again incubated for 5 min at 37°C. One milliliter of 25% TCA
solution is then added to stop the reaction. To quantify the ~-naphthy-
lamine released, 1 mL of this solution is mixed with 1 mL of 0.1%
NaNO 3. After 3 min, 1 mL of a 0.5% ammonium sulfamate solution is
added to destroy excess nitrite. After 5 min, 2 mL of 0.05 % N- 1-naphthy-
lethylenediamine dihydrochloride solution is added, and the solution