Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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METALLOPROTEASES 237
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CHAPTER 15
AMINOPEPTIDASES
YOLANDA SANZ
∗
Departamento de Ciencia de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos,
CSIC, Paterna, Valencia, Spain
∗
yolsanz@iata.csic.es
1. INTRODUCTION
Aminopeptidases hydrolyse peptide bonds at the N-terminus of proteins and
polypeptides whereas carboxypeptidases hydrolyse peptide bonds at the C-terminus.
Omega peptidase is an additional term referring to special types of aminopep-
tidases and carboxypeptidases that are capable of removing terminal residues
lacking a free -amino or -carboxyl group, or include linkages other than
the -peptide type (e.g. pyroglutamyl peptidases; McDonald and Barret, 1986).
Aminopeptidases can be subdivided into three groups: aminopeptidases in the
strict sense which hydrolyse the first peptide bond in a polypeptide chain with
the release of a single amino acid residue (aminoacyl- and iminoacyl peptidases
[EC 3.4.11]); those that remove dipeptides or tripeptides (dipeptidyl- and tripep-
tidyl peptidases [EC 3.4.14]) from polypeptide chains; and those which only
hydrolyse di- or tripeptides (dipeptidases [EC 3.4.15] and tripeptidases [EC
3.4.14.4]) (Sanderink et al., 1988). Aminopeptidases are widely distributed among
bacteria, fungi, plants and mammals (Gonzales and Robert-Baudouy, 1996; Sanz
et al., 2002; Tu et al., 2003; Barret et al., 2004). Theses enzymes are located
in different subcellular compartments including the cytoplasm, lysosomes and
membranes, and can also be secreted into the extracellular medium. Based on
catalytic mechanism, most of the aminopeptidases are metallo-enzymes but cysteine
and serine peptidases are also included in this group. Though some aminopep-
tidases are monomeric, most show multimeric structures particularly those from
eukaryotic organisms (McDonald and Barret, 1986; Jones, 1991; Lowther and
Matthews, 2002). The three-dimensional structures of some aminopeptidases have
been solved, contributing to the understanding of their catalytic mechanism and
243
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 243–260.
© 2007 Springer.
244 SANZ
functions (Kim et al., 1993; Bazan et al., 1994; Joshua-Tor et al., 1995; Lowther
and Matthews, 2002). Aminopeptidases appear to act in concert with other pepti-
dases to complete diverse proteolytic pathways. Thus, these enzymes can efficiently
retrieve amino acids from dietary proteins and endogenous proteins degraded
during protein turnover, thereby covering nutritional as well as other biological
roles including protein maturation, hormone level regulation and cell-cycle control
(McDonald and Barret, 1986; Christensen et al., 1999). Many of the mammalian
enzymes play important functions in cellular processes involved in health and
disease and, as a consequence, constitute targets for the pharmaceutical industry
(Scornik and Botbol, 2001; Holz et al., 2003; Rigolet et al., 2005; Inguimbert
et al., 2005). Some aminopeptidases are also of great interest for their biotech-
nological and agro-industrial applications (Seppo et al., 2003; FitzGerald and
O’Cuinn, 2006).
2. CLASSIFICATION AND NOMENCLATURE
OF AMINOPEPTIDASES
Aminopeptidases have been classified on the basis of their substrate specificity
(broad or narrow), catalytic mechanism (metallo-, cysteine, and serine peptidases)
and molecular structure (Gonzales and Robert-Baudouy, 1996; Barret et al., 2004;
Rawling et al., this volume). The nomenclature of many (aminoacyl or iminoacyl
peptidases) has been determined by their preferences or requirements for a particular
N-terminal amino acid. Thus, an enzyme that for instance showed its highest rate of
hydrolysis on N-terminal methionyl bonds was named methionyl aminopeptidase
or aminopeptidase M. In an attempt to avoid ambiguity, the subcellular location
(membrane, microsomal or cytosolic) has also been used to name aminopeptidases
having similar specificities. In the cases of di- and tripeptidases their names have
been based on substrate size requirements. In addition, the names of dipeptidyl
(DPP) and tripeptidyl peptidases (TPP) were followed by a Roman number to differ-
entiate between the various types described and this numbering convention has been
retained. In the nomenclature of peptidases identified in lactic acid bacteria the term
‘Pep’ is used when the corresponding peptidase gene sequence is known, followed
by a capital letter indicating the specificity and homology to other known pepti-
dases, e.g. PepN for the homologue of aminopeptidase N (Tan et al., 1993). Never-
theless, alternative names and abbreviations often appear in the literature. Recently,
the MEROPS peptidase information database (http://www.merops.sanger.ac.uk;
see chapter by Rawlings et al. in this volume) created a hierarchical structure-
based classification of peptidases into families and clans. Members of a family
are homologues, and families that are thought to be homologous are grouped
together into clans. Clans consist of families of peptidases that share a single
evolutionary origin, evidenced by similarities in their tertiary structures and/or the
order of catalytic-site residues and common sequence motifs around the catalytic
residues.
AMINOPEPTIDASES 245
3. ANIMOPEPTIDASE TYPES AND FUNCTIONS
3.1. Mammalian Aminopeptidases
Aminopeptidases were among the first proteases to be discovered in mammalian
tissues and a large number have already been characterized (Barret et al., 2004). The
best characterized mammalian aminopeptidases and their biochemical properties
are shown in Table 1. On the basis of specificity they can be divided into different
groups: (i) aminopeptidases of broad specificity (e.g. leucyl aminopeptidase, and
membrane and cytosol alanyl aminopeptidases); (ii) aminopeptidases of narrow
specificity with preference for basic amino acid residues (aminopeptidase B), acid
amino acid residues (glutamyl and aspartyl aminopeptidases), cysteine (cystinyl
aminopeptidase), methionine (methionyl aminopeptidase), or bonds containing
proline (prolyl aminopeptidase and aminopeptidase P); and (iii) dipeptidyl pepti-
dases (DPP I, II, III, and IV) and tripeptidyl peptidases (TPP I and II) that release di-
and tripeptides; respectively (Cunningham et al., 1997; Sanz et al., 2002; Albiston
Table 1. Main types and properties of mammalian aminopeptidases
Enzyme EC number Catalytic type Specificity Family
Aminopeptidase (AP) X−⇓−Y −Z
n
Leucyl AP 3.4.11.1 Metallo X = Leu M17
Membrane alanyl AP 3.4.11.2 Metallo X = Ala,
Phe, Tyr, Leu
M1
Cytosol
alanyl AP
3.4.11.14 Metallo X = Ala M1
Aminopeptidase B 3.4.11.6 Metallo X = Arg, Lys M1
Glutamyl AP 3.4.11.7 Metallo X = Glu, Asp M1
Cystinyl AP 3.4.11.3 Metallo X = Cys M1
Methionyl AP 3.4.11.18 Metallo X = Met M24A
Aminopeptidase P 3.4.11.9 Metallo X and Y = Pro M24B
Prolyl AP (PIP) 3.4.11.5 Serine X = Pro S33
Bleomycin hydrolase 3.4.22.40 Cysteine X = Met, Leu, Ala
Bleomycin peptide
C1B
Dipeptidyl peptidases (DPP) X-Y-⇓-(Z)n
DPP I 3.4.14.1 Cysteine X = Arg or Lys,
YorZ= Pro
C1
DPP II 3.4.14.2 Serine Y = Ala or Pro S28
DPP III 3.4.14.4 Metallo X-Y = Arg-Arg M49
DPP IV 3.4.14.5 Serine Y = Pro S9B
Tripeptidyl peptidases
(TPP)
X-Y-T-⇓-(Z)n
TPP I 3.4.14. 9 Serine Gly-Pro-T =
hydrophobic
S53
TPP II 3.4.14.10 Serine Ala-Ala-Phe
TorZ= Pro
S8
246 SANZ
et al., 2004). The characteristics of the main aminopeptidases of broad and narrow
specificities and proline-specific peptidases are briefly reviewed.
3.1.1. Mammalian aminopeptidases of broad specificity
Leucyl aminopeptidase (LAP) is a ubiquitous enzyme that has also been referred
to as cytosol aminopeptidase and leucine aminopeptidase. It was the first cytosolic
aminopeptidase to be identified (Linderstrom-Lang, 1929). LAP preferentially
releases Leu located as the N-terminal residue of peptides, and can also release
other amino acids including Pro but not Arg or Lys. This enzyme is a hexamer
of identical chains and has a molecular mass of 324-360 (Kohno et al., 1986).
Human LAP is involved in the breakdown of the peptide products of intracellular
proteinases and is one of the enzymes that trims proteasome-produced peptides for
presentation by the major histocompatibility complex class I molecules. Expression
of the encoding gene is promoted by interferon gamma (Beninga et al., 1998).
Membrane alanyl aminopeptidase has also been referred to as aminopeptidase M
due to its membrane localization since there is a cytosolic counterpart, and also as
aminopeptidase N due to its preference for neutral amino acids. The amino acid
residue preferentially released is Ala, but most amino acids including Pro may
be hydrolysed by this enzyme. When a terminal hydrophobic residue is followed
by Pro, the two may be released as an intact X-Pro dipeptide (McDonald and
Barret, 1986). In most species the native enzyme is a homodimer with a molecular
mass of 280-300 and is glycosylated. The mammalian enzyme plays a role in the
final digestion of peptides generated from proteins by gastric and pancreatic protease
hydrolysis. It is also important for the inactivation in the kidney of blood-borne
peptides such as enkephalins and the neuropeptide ‘substance P’. Furthermore, it is a
regulator of IL-8 bioavailability in the endometrium and therefore may contribute to
the regulation of angiogenesis. This aminopeptidase is also the myeloid leukaemia
marker CD13 and serves as a receptor for human coronavirus (Shimizu et al., 2002;
Albiston et al., 2004).
3.1.2. Mammalian aminopeptidases of narrow specificity
Glutamyl aminopeptidase is also referred to as aminopeptidase A, angiotensinase A
and aspartate aminopeptidase. It releases N-terminal Glu (and to a lesser extent
Asp) from a peptide. It is generally a membrane-bound enzyme involved in the
formation of the brain heptapeptide angiotensin III which exerts a tonic stimulatory
effect on the central control of blood pressure and is a regulator of blood vessel
formation (Fournie-Zaluski et al., 2004.).
Methionyl aminopeptidase has also been named methionine aminopeptidase and
peptidase M. It releases N-terminal amino acids, preferentially methionine, from
peptides but only when the second residue is small and uncharged. In eukaryotes,
two types of methionyl aminopeptidases exist due to protein synthesis occurring
in the mitochondria (type I) and in the cytoplasm (type II). The type I peptidase
is similar to the bacterial methionyl aminopeptidase whereas type II resembles the
enzyme from archaea (Arfin et al., 1995). The mammalian enzymes are involved in