320 Sweeney and WalkerCONH 2
CH2
~H2 I
H2NCHCOLH2.C CH 2
I
O~.NJCHCO ~ + NH3Fig. 1. The cyclization of N-terminal glutamine to pyroglutamic (pyrrolidone
carboxylic) acid.therefore, the peptide or protein is not amenable to sequence determi-
nation, unless the pyroglutamyl derivative is removed by pyroglutamate
aminopeptidase (11,12). The enzyme was first purified from Pseudomo-
nasfluoresens (12), but nowadays the calf liver enzyme is used, and
it is this enzyme that we describe.
Aminopeptidase M, a zinc-containing metalloprotease, from swine
kidney microsomes (13-16) removes amino acids sequentially from
the N-terminals of peptides and proteins. It, therefore, has some use in
the determination of N-terminal sequence data, although the Edman
degradation would probably be the method of choice for most work-
ers. Aminopeptidase M is more frequently used in the preparation of
peptide and protein hydrolysates for amino acid analysis. Tradition-
ally, peptides and proteins are hydrolyzed in 6N HC1, but this approach
results in the total loss of tryptophan, partial loss (5-10%) of serine
and threonine, and hydrolysis of asparagine and glutamine to the cor-
responding acids. The use of enzymes to produce a peptide/protein
hydrolysate overcomes these problems. Although aminopeptidase
M is capable of cleaving all possible peptide bonds, in practice, the
X--Pro bond is not completely cleaved and the dipeptide X--Pro is
released (17).
Aminopeptidase M therefore tends to be used in conjunction with
our third enzyme, prolidase. Prolidase, a manganese-containing
metalloprotease, has been purified from a number of sources, but the
porcine kidney enzyme is generally used. It is more correctly a highly
specific imidopeptidase, since it cleaves the dipeptide X--Pro or X--
Hypro (18). The amino acid sequence and gene location of human
prolidase have been elucidated (19), and active site modeling studies
of the enzyme have been performed (20).