characterization compared to dipeptidyl peptidase IV. Biochem. J.

396(2), 391�399.

[60] Lankas, G., Leiting, B., Roy, R., Eiermann, G., Beconi, M.,

Biftu, T., Chan, C., Edmondson, S., Feeney, W., He, H.,

Ippolito, D., Kim, D., Lyons, K., Ok, H., Patel, R., Petrov, A.,

Pryor, K., Qian, X., Reigle, L., Woods, A., Wu, J., Zaller, D.,

Zhang, X., Zhu, L., Weber, A., Thornberry, N. (2005). Dipeptidyl

peptidase IV inhibition for the treatment of type 2 diabetes �Potential importance of selectivity over dipeptidyl peptidases 8 and 9.

Diabetes 54(10), 2988�2994.

[61] Yu, D.M.T., Yao, T.-W., Chowdhury, S., Nadvi, N.A.,

Osborne, B., Church, W.B., McCaughan, G.W., Gorrell, M.D.

(2010). The dipeptidyl peptidase IV family in cancer and cell biol-

ogy. FEBS J. 277, 1126�1144.

Mark D. GorrellCentenary Institute, Sydney Medical School, University of Sydney, NSW 2006, Australia. Email: m.gorrell@centenary.usyd.edu.au

John E. ParkNBE Discovery, Boehringer Ingelheim Pharma KG, Birkendorferstr. 65, 88397 Biberach, Germany. Email: john.park@bc.boehringer-ingelheim.com

Handbook of Proteolytic Enzymes, 3rd Edn © 2013 Elsevier Ltd. All rights reserved.

ISBN: 978-0-12-382219-2 DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00750-X

Chapter 751

Acylaminoacyl-Peptidase

DATABANKS

MEROPS name: acylaminoacyl-peptidase

MEROPS classification: clan SC, family S9, subfamily

S9C, peptidase S09.004

IUBMB: EC 3.4.19.1 (BRENDA)

Species distribution: superkingdom Eukaryota

Reference sequence from: Homo sapiens (UniProt:

P13798)

Name and History

Acylaminoacyl-peptidase (EC 3.4.19.1) has also been

referred to by the names acylpeptide hydrolase (APH)

[1�3], acylamino acid-releasing enzyme [4,5], and acy-

laminoacyl peptide hydrolase [6].

Activity and Specificity

Acylaminoacyl-peptidase catalyzes the removal of an

N-acylated amino acid from a blocked peptide. The pro-

ducts of the reaction are an acyl amino acid and a pep-

tide with a free N-terminus shortened by one amino

acid. The enzyme acts on a variety of peptides with dif-

ferent N-terminal acyl groups, including acetyl, chloro-

acetyl, formyl and carbamyl [7]. The optimum length of

the blocked peptide substrate is 2�3 amino acids, but

larger substrates are also cleaved, at slower rates [8].

For instance, the blocked 13 residue peptide α-melano-

cyte stimulating hormone (αMSH) is a substrate [7]. On

the other hand, N-terminally blocked proteins are not

substrates for the enzyme. The enzyme is active over a

wide pH range, depending on the substrate used [2].

For example, with Ac-GlukNHPhNO2, the pH optimum

is around 6, but for Ac-AlakNHPhNO2, it is near pH

8.4. For most acetylated peptide substrates the pH opti-

mum is in the range 7.3�7.6. Esters such as p-nitrophe-

nyl propionate and α-naphthyl butyrate are also

efficiently hydrolyzed by the enzyme. In addition to its

omega peptidase activity, acylaminoacyl-peptidase may

also have endopeptidase activities. Thus, Fujino et al.

[9] described an ‘oxidized protein hydrolase’ in human

erythrocyte cytosol. This enzyme, which preferentially

degrades oxidatively damaged proteins, was claimed to

be acylaminoacyl-peptidase. Scaloni et al. found that

trypsin selectively cleaves the enzyme into 22 and

53 kDa fragments that remain attached in the native

enzyme with retention of activity [10]. Senthilkumar

et al. [11] found that cleavage between amino acid resi-

dues 203 and 204 of acylaminoacyl-peptidase occurs in

bovine lens in vivo, and that it results in the formation

of a 55 kDa truncated enzyme that has endopeptidase

activity. The truncated enzyme cleaved β2-crystallininto protein fragments of 10�26 kDa.

3401Clan SC � S9 | 751. Acylaminoacyl-Peptidase

Structural Chemistry

The enzyme is composed of four identical subunits, each

containing 732 amino acids and a blocked N-terminus for

a total molecular mass of about 300 kDa [1�4] with an

isoionic point of 4.1 [12]. The enzyme is inhibited by sev-

eral types of reagents including DFP, PCMB [13],

diethylpyrocarbonate and some heavy metals, such as

Hg21, Zn21 and Cd21 [1]. The inhibition by DFP is due

to modification at Ser597, and Ac-Leu-CH2Cl inactivates

the enzyme by reaction at the active-site His707 [13].

These sites constitute two parts of the catalytic triad

[13,14]. It is notable that the His residue of the catalytic

triad is located closest to the C-terminus of the protein.

Mitta et al. [15] confirmed the identification of the resi-

dues of the catalytic triad.

Initial crystal structure studies of the human enzyme

showed it to have a canonical α/β hydrolase fold domain

[16]. The homologous enzyme was crystallized from the

archaeon Aeropyrum pernix K1 (deemed apAPH), which

helped to confirm its membership in the family of prolyl

oligopeptidase serine proteases and its α/β fold domain

as well as to reveal a regular seven-bladed β-propellerN-terminal domain [17]. In addition to the catalytic triad,

several other residues were shown to play roles outside

the catalytic domain, including Glu88 in apAPH, which

neutralizes a charge for enzymatic activity and creates a

thermodynamically stable salt bridge with Arg526, a part

of the triad in the archaeon enzyme [18]. Studies in puri-

fied porcine enzyme demonstrated the importance of resi-

due His507 in stabilizing the active site conformation

[19]. The enzyme has also been characterized in plants

where it was shown to have similar composition to the

mammalian enzyme as well as comparable response to

inhibitors [20].

Preparation

The enzyme has been purified to homogeneity from

human erythrocytes [13,21], cattle liver [1], rabbit muscle

[6], and porcine liver [22]. However, activity has been

detected in practically all tissues examined [4]. APH from

various sources co-purifies with thimet oligopeptidase

(Chapter 101).

Biological Aspects

The true biological function of this enzyme is not known,

but several biological peptide substrates have been identi-

fied. Although it does not act on acetylated protein sub-

strates, it does hydrolyze small N-terminally blocked

peptides, some of which are bioactive peptides. It could

conceivably act on the exposed N-terminus of nascent

polypeptide chains. In this regard, some tumor cells, in

which 80% of all proteins are blocked at their N-termini,

do not contain this enzyme [23]. In small-cell lung carci-

noma cell lines, in which the locus on the short arm of

chromosome 3 containing the gene encoding this enzyme

undergoes deletions, the enzyme is not expressed [23]. In

such cells an accumulation of unhydrolyzed acetylated

peptide growth factors could lead to cell proliferation, but

this hypothesis requires verification. The enzyme was

also shown to degrade monomeric and oligomeric amy-

loid-beta peptides in vitro, with preference to the more

toxic oligomeric species. In addition, its expression level

was upregulated in human Alzheimer’s disease brains as

compared to age-matched controls [24,25]. Other major

endopeptidases that cleave amyloid-beta peptides are

neprilysin (Chapter 127) and insulin degrading enzyme or

insulysin (Chapter 318).

Because of the inhibitory specificity of several organo-

phosphate (OP) compounds to the enzyme, it has been pro-

posed to use blood APH activity as a sensitive marker for

exposure to OP compounds [26,27]. Ironically, organopho-

sphates are also prime therapeutic candidates for the treat-

ment of Alzheimer’s disease as they have been shown to

increase synaptic plasticity and enhance long-term potenti-

ation in rat hippocampal slices via an inhibition of the

enzyme at low doses, leading to cognitive enhancement

[28,29].

References[1] Gade, W., Brown, J.L. (1978). Purification and partial characteriza-

tion of α-N-acylpeptide hydrolase from bovine liver. J. Biol.

Chem. 253, 5012�5018.

[2] Jones, W.M., Manning, J.M. (1985). Acylpeptide hydrolase activity

from erythrocytes. Biochem. Biophys. Res. Commun. 126, 933�940.

[3] Kobayashi, K., Lin, L.-W., Yeadon, J.E., Klickstein, L.B.,

Smith, J.A. (1989). Cloning and sequence analysis of a rat liver

cDNA encoding acylpeptide hydrolase. J. Biol. Chem. 264,

8892�8899.

[4] Tsunasawa, S., Narita, K., Ogata, K. (1975). Purification and prop-

erties of acylamino acid-releasing enzyme from rat liver. J.

Biochem. 77, 89�102.

[5] Mitta, M., Asada, K., Uchimura, Y., Kimizuka, F., Kato, I.,

Sakiyama, F., Tsunasawa, S. (1989). The primary structure of por-

cine liver acylamino acid-releasing enzyme deduced from cDNA

sequences. J. Biochem. 106, 548�551.

[6] Radhakrishna, G., Wold, F. (1989). Purification and characteriza-

tion of an N-acylaminoacyl-peptide hydrolase from rabbit muscle.

J. Biol. Chem. 264, 11076�11081.

[7] Jones, W.M., Manning, L.R., Manning, J.M. (1986). Enzymic

cleavage of the blocked amino terminal residues of peptides.

Biochem. Biophys. Res. Commun. 139, 244�250.

[8] Jones, W.M., Scaloni, A., Bossa, F., Popowicz, A.M.,

Schneewind, O., Manning, J.M. (1991). Genetic relationship

between acylpeptide hydrolase and acylase, two hydrolytic

enzymes with similar binding but different catalytic specificities.

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[9] Fujino, T., Watanabe, K., Beppu, M., Kikugawa, K., Yasuda, H.

(2000). Identification of oxidized protein hydrolase of human ery-

throcytes as acylpeptide hydrolase. Biochim. Biophys. Acta Protein

Struct. Mol. Enzymol. 1478, 102�112.

[10] Scaloni, A., Ingallinella, P., Andolfo, A., Jones, W., Marino, G.,

Manning, J.M. (1999). Structural investigations on human erythro-

cyte acylpeptide hydrolase by mass spectrometric procedures.

J. Protein Chem. 18, 349�360.

[11] Senthilkumar, R., Reddy, P.N., Sharma, K.K. (2001). Studies on

trypsin-modified bovine and human lens acylpeptide hydrolase.

Exp. Eye Res. 72, 301�310.

[12] Scaloni, A., Barra, D., Jones, W.M., Manning, J.M. (1994). Human

acylpeptide hydrolase studies on its thiol groups and mechanism of

action. J. Biol. Chem. 269, 15076�15084.

[13] Scaloni, A., Jones, W.M., Barra, D., Pospischil, M., Sassa, S.,

Popowicz, A., Manning, L.R., Schneewind, O., Manning, J.M.

(1992). Acylpeptide hydrolase: inhibitors and some active site resi-

dues of the human enzyme. J. Biol. Chem. 267, 3811�3818.

[14] Rawlings, N.D., Polgar, L., Barrett, A.J. (1991). A new family of

serine-type peptidases related to prolyl oligopeptidase. Biochem. J.

279, 907�908.

[15] Mitta, M., Miyagi, M., Kato, I., Tsunasawa, S. (1998).

Identification of the catalytic triad residues of porcine liver acyla-

mino acid-releasing enzyme. J. Biochem. (Tokyo) 123, 924�931.

[16] Feese, M., Scaloni, A., Jones, W.M., Manning, J.M., Remington, S.J.

(1993). Crystallization and preliminary X-ray studies of human eryth-

rocyte acylpeptide hydrolase. J. Mol. Biol. 223, 546�549.

[17] Bartlam, M., Wang, G., Yang, H., Gao, R., Zhao, X., Xie, G.,

Cao, S., Feng, Y., Rao, Z. (2004). Crystal structure of an acylpep-

tide hydrolase/esterase from Aeropyrum pernix K1. Structure 12,

1481�1488.

[18] Yang, G., Bai, A., Gao, L., Zhang, Z., Zheng, B., Feng, Y. (2009).

Glu88 in the non-catalytic domain of acylpeptide hydrolase plays

dual roles: charge neutralization for enzymatic activity and forma-

tion of salt bridge for thermodynamic stability. Biochim. Biophys.

Acta 1794, 94�102.

[19] Kiss, A.L., Szeltner, Z., Fulop, V., Polgar, L. (2004). His507 of

acylaminoacyl peptidase stabilizes the active site conformation, not

the catalytic intermediate. FEBS Lett. 571, 17�20.

[20] Yamauchi, Y., Ejiri, Y., Toyoda, Y., Tanaka, K. (2003).

Identification and biochemical characterization of plant acylamino

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[21] Jones, W.M., Scaloni, A., Manning, J.M. (1994). Acylaminoacyl

peptidase. Methods Enzymol. 244, 227�231.

[22] Wright, H., Kiss, A.L., Szeltner, Z., Polgar, L., Fulop, V. (2005).

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[23] Scaloni, A., Jones, W., Pospischil, M., Sassa, S., Schneewind, O.,

Popowicz, A.M., Bossa, F., Graziano, S.L., Manning, J.M. (1992).

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cell lines. J. Lab. Clin. Med. 120, 546�552.

[24] Yamin, R., Bagchi, S., Hildebrant, R., Scaloni, A., Widom, R.L.,

Abraham, C.R. (2007). Acyl peptide hydrolase, a serine proteinase

isolated from conditioned medium of neuroblastoma cells,

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[25] Yamin, R., Zhao, C., O’Connor, P.B., McKee, A.C.,

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[27] Kim, J.H., Stevens, R.C., Maccoss, M.J., Goodlett, D.R.,

Scherl, A., Richter, R.J., Suzuki, S.M., Furlong, C.E. (2010).

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phorus exposures in humans. Adv. Exp. Med. Biol. 660, 61�71.

[28] Richards, P.G., Johnson, M.K., Ray, D.E. (2000). Identification of

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phosphorus compounds and a potential target for cognitive enhanc-

ing drugs. Mol. Pharmacol. 58, 577�583.

[29] Olmos, C., Sandoval, R., Rozas, C., Navarro, S., Wyneken, U.,

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Carmela R. AbrahamDepartment of Biochemistry and Program in Molecular Medicine, Boston University School of Medicine, 72 E. Concord Street, K304, Boston, MA

02118, USA. Email: cabraham@bu.edu

Michael W. NagleDepartment of Biochemistry and Program in Molecular Medicine, Boston University School of Medicine, 72 E. Concord Street, K304, Boston, MA

02118, USA.

This article is a revision of the previous edition article by James M. Manning, Volume 2, pp. 1917�1919, r 2004, Elsevier Ltd.

Handbook of Proteolytic Enzymes, 3rd Edn © 2013 Elsevier Ltd. All rights reserved.

ISBN: 978-0-12-382219-2 DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00751-1

3403Clan SC � S9 | 751. Acylaminoacyl-Peptidase