Protein N-terminal processing: Substrate specificity of escherichia coli and human methionine aminopeptidases

Article abstract of DOI:10.1021/bi1005464

Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of removal of methionyl from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1′ residue, but their specificity at positions P2′ and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1′ position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently 2 orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2′-P5′, all three MetAPs have broad specificity but are poorly active toward peptides containing a proline at the P2′ position. In addition, the human MetAPs disfavor acidic residues at the P2′-P5′ positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins.

Full text of DOI:10.1021/bi1005464

5588 Biochemistry 2010, 49, 5588–5599
DOI: 10.1021/bi1005464
Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and
Human Methionine Aminopeptidases†
Qing Xiao,‡ Feiran Zhang,§ Benjamin A. Nacev,§ Jun O. Liu,§ and Dehua Pei*,‡
‡Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, and §Department of
Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205
Received April 11, 2010; Revised Manuscript Received June 1, 2010
ABSTRACT: Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine
from newly synthesized polypeptides. The extent of removal of methionyl from a protein is dictated by its
N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains
(e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P10 residue, but their specificity at positions P20 and beyond
remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1,
and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic
analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require
small residues at the P10 position, they have differential tolerance for Val and Thr at this position. The catalytic
activity of human MetAP2 toward Met-Val peptides is consistently 2 orders of magnitude higher than that of
MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-
Thr sequences in vivo. At positions P20-P50, all three MetAPs have broad specificity but are poorly active toward
peptides containing a proline at the P20 position. In addition, the human MetAPs disfavor acidic residues at the
P20-P50 positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the
N-terminal processing of E. coli and human proteins.
Ribosomal protein synthesis is universally initiated with methio-
nine (in eukaryotic cytoplasm) or N-formylmethionine (in prokar-
yotes, mitochondria, and chloroplasts). During protein matura-
tion, the N-formyl group is removed by peptide deformylase,
leaving methionine with a free NH2 group (1). Subsequently, the
initiator methionine is removed from many but not all of the pro-
teins by methionine aminopeptidase(s) (MetAPs).1 For example, in
a cytosolic extract of Escherichia coli, only 40% of the polypeptides
retain the initiator methionine, whereas the majority of the poly-
peptides display alanine, serine, or threonine at their N-termini (2).
There are two types of MetAPs, MetAP1 and MetAP2. While
eubacteria have only MetAP1 and archaea have only MetAP2,
eukaryotes, including yeast, have both MetAP1 and MetAP2 in the
cytoplasm and MetAP1D in the mitochondria (3). MetAP activities
are essential for survival in both bacteria and yeast (4-6). As a
result, there has been a renewed interest in designing specific inhi-
bitors against bacterial MetAP1 as novel antibiotics (7-10). We
and others have previously discovered that MetAP2 is the molec-
ular target of fumagillin, a fungal metabolite and potent inhibitor of
angiogenesis, validating MetAP2 as a novel target for anticancer
treatment (11, 12). In fact, a semisynthetic analogue of fumagillin,
TNP-470, has entered phase II clinical trials as an anticancer
agent (13). TNP-470 preferentially inhibits endothelial cell growth
in tumor vasculature by arresting the cell cycle in the late G1
phase (14, 15). Presumably, inhibition of MetAP2 by TNP-470
prevents the removal of methionine from one or more critical
proteins and therefore their maturation. However, the identity of
these MetAP2-specific substrates is currently unknown.
N-Terminal methionine removal is a cotranslational process
and occurs as soon as a polypeptide emerges from the ribo-
some (16). Previous studies indicate that the fate of the N-terminal
methionine is dictated by the substrate specificities of MetAPs,
which require Met as the P1 residue and amino acids with small
side chains as the P10 residue (e.g., Ala, Gly, Pro, Ser, Thr, or
Val) (17-24). Amino acid residues at positions P20 and beyond
also play a role. For example, a hemoglobin variant with a proline
at position 3 (P20) retains the initiator methionine in vivo, whereas
mutation of the proline to Arg or Gln leads to complete cleavage
of the methionine (20). Chang et al. (21) reported a 2-fold diffe-
rence in the activity of purified yeast MetAP1 toward Met-Ala-
Ile-Pro-Glu versus Met-Ala-Ile-Pro-Ser, which differ only at the
P40 position. These studies clearly demonstrate the existence of
sequence specificity by MetAPs. However, because these studies
employed the individual synthesis and activity assay of each pep-
tide, the sequences that have been tested represent only a small
fraction of the possible protein N-terminal sequence space and are
generally limited to the examination of the specificity at the P1,
P10, and occasionally P20 positions. One recent study by Frottin
et al. has generated a more complete specificity profile of E. coli
MetAP1 and Pyrococcus furiosus MetAP2, via the synthesis and
†This work was supported by grants from the National Institutes of
Health (NIH) (GM062820, CA132855, and CA078743). B.A.N. was
supported by NIH Medical Scientist Training Program Grant
T32GM07309.
*To whom correspondence should be addressed: Department of Che-
mistry, The Ohio State University, 100 W. 18th Ave., Columbus, OH 43210.
Telephone: (614) 688-4068. Fax: (614) 292-1532. E-mail: pei.3@osu.edu.
1Abbreviations: Abu (U), 2-aminobutyrate; Dabcyl-β-Ala-OSu, N-hydro-
xylsuccinimidyl 3-{4-[4-(dimethylamino)benzoylamino}propionate; Dabcyl-
OSu, N-{4-[40-(dimethylamino)phenylazo]benzoyloxy}succinimide; Dde,
N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl; EDC, 1-ethyl-3-(3-di-
methylaminopropyl)carbodiimide; Fmoc-OSu, N-(9-fluorenylmethoxycar-
bonyloxy)succinimide; HBTU, O-benzotriazole-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole hydrate;
MetAP, methionine aminopeptidase; Nle (M), norleucine; OBOC, one-
bead-one-compound; PCR, polymerase chain reaction; PED-MS, partial
Edman degradation mass spectrometry; PEGA, amino polyethylene
glycol; PGM1, phosphoglucomutase; PITC, phenyl isothiocyanate;
TXNL1, thioredoxin-like protein 1.
r
pubs.acs.org/Biochemistry
Published on Web 06/03/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 26, 2010 5589
assay of ∼120 short peptides (25). To the best of our knowledge,
no systematic study has been performed for any eukaryotic
MetAPs. A potential approach to systematically determining
the substrate specificity of a MetAP is to screen it against a
combinatorial peptide library. This, however, has not been possi-
ble because of the technical difficulty in differentiating reaction
products from unreacted substrates, which are both peptides with
free N-termini. Herein, we report a novel peptide library screening
strategy and its application to determine the sequence specificity
of E. coli MetAP1 and human MetAP1 and MetAP2. The speci-
ficity information should be useful in designing specific MetAP2
inhibitors as anticancer agents and compounds specific for the
bacterial MetAP1 as antibacterial agents.
coupled twice with 4 equiv of a different Fmoc amino acid with
HBTU, HOBt, and N-methylmorpholine (NMM) for 2 h at room
temperature. To differentiate isobaric amino acids during MS
analysis, 5% (mol/mol) CD3CO2D was added to the coupling
reaction mixture of Leu, whereas 5% CH3CD2CO2D was added to
the coupling reaction mixture of norleucine (27). After the last
random residue had been coupled, the resin was pooled into three
sublibraries according to the identity of the last random residue.
Sublibraries I and II contained Ser and Thr as the N-terminal
residues, respectively, while sublibrary III contained an equimolar
mixture of the other 16
L-R-amino acids excluding Ser and
Thr. The three sublibraries were separately coupled with 4 equiv
of Fmoc-Met-OH and deprotected by treatment with the modi-
fied reagent K (79:7.5:5:5:2.5:1 TFA/phenole/H2O/thioanisole/
ethanedithiol/anisole). The resin-bound sublibraries were washed
exhaustively with CH2Cl2 and DMF, suspended in DMF, and
stored under an argon atmosphere at 4 °C.
MATERIALS AND METHODS
Materials and General Methods. 1H and 13C NMR spectra
were recorded on a Bruker 400 DPX spectrometer at 400
N-Hydroxylsuccinimidyl 3-{4-[4-(Dimethylamino)phe-
nylazo]benzoylamino}propionate (Dabcyl-β-Ala-OSu). To
a 100 mL dry flask were added Dabcyl-OSu (0.366 mg, 1 mmol),
DMF (25 mL), triethylamine (0.42 mL, 3.0 mmol), and β-alanine
(0.091 g, 1.02 mmol). The mixture was stirred at room tempera-
ture for 18 h. The solvent was removed under vacuum, and the
solid residue was washed with H2O and then dried via azeotrope
with toluene to give 0.322 g (88% yield) of a crude product. The
crude product (0.322 g, 0.946 mmol) was dissolved in DMF
(15 mL), to which N-hydroxysuccinimide (0.163 g, 1.42 mmol)
and EDCI (0.272 g, 1.42 mmol) had been added. The mixture was
stirred overnight, and the solvent was removed under vacuum.
The solid residue was dissolved in dichloromethane, washed with
H2O, dried over Na2SO4, and concentrated to give a red solid
(0.372 g, 90% yield): 1H NMR (250 MHz, CDCl3) δ 7.96-7.86
(m, 6H), 6.77 (d, 2H, J=9.3 Hz), 3.92 (m, 2H), 3.11 (s, 6H), 3.00-
2.95 (m, 2H), 2.89 (s, 4H); HRMS (ESI) calcd for C22H24N5O5
(Mþ þ H) 438.1772, found 438.1764.
and 100 MHz, respectively. Fmoc-protected L-amino acids were
purchased from Advanced ChemTech (Louisville, KY), Peptides
International (Louisville, KY), or NovaBiochem (La Jolla, CA).
O-Benzotriazole-N,N,N0,N0-tetramethyluronium hexafluoropho-
sphate (HBTU) and 1-hydroxybenzotriazole hydrate (HOBt) were
from Peptides International. All solvents and other chemical re-
agents were obtained from Aldrich, Fisher Scientific (Pittsburgh,
PA), or VWR (West Chester, PA) and were used without further
purification unless noted otherwise. N-(9-Fluorenylmethoxycarbo-
nyloxy)succinimide (Fmoc-OSu) was from Advanced ChemTech.
Phenyl isothiocyanate (PITC) was purchased in 1 mL sealed
ampules from Sigma-Aldrich, and a freshly opened ampule was
used in each experiment. N-{4-[40-(Dimethylamino)phenylazo]-
benzoyloxy}succinimide (Dabcyl-OSu) was from ABD Bioquest
(Sunnyvale, CA). Amino polyethylene glycol (PEGA) resin
(0.4 mmol/g, 150-300 μm in water) was from Peptides Interna-
tional. Clear amide resin (90 μm, 0.49 mmol/g) was from Advanced
ChemTech. R-Cyano-4-hydroxycinnamic acid (R-CCA) was pur-
chased from Sigma and recrystallized prior to use. E. coli MetAP1
was purified from an E. coli strain overproducing the enzyme as
previously described (26). Recombinant human MetAP1 and Met-
AP2 were expressed and purified as previously described (11, 26).
Anti-Flag, anti-PGM1, and anti-TXNL1 antibodies were pur-
chased from Sigma or Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA).
Synthesis of the MetAP Library. The peptide library was
synthesized on 2.0 g of PEGA resin (0.4 mmol/g, 150-300 μm in
water). All of the manipulations were performed at room tem-
perature unless otherwise noted. Synthesis of the N-1-(4,4-dimeth-
yl-2,6-dioxocyclohexylidene)ethyl (Dde) linker began with acyla-
tion of the resin-bound amine with glutaric anhydride (3.0 equiv) in
15 mL of CH2Cl2 containing diisopropylethylamine (1.1 equiv).
After 3 h, the resin was washed with CH2Cl2 and the newly formed
carboxylic acid was treated with 15 mL of CH2Cl2 containing
5,5-dimethylcyclohexanedione (4.0 equiv), 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide (EDCI, 2 equiv), and N,N-diamino-
pyridine (2 equiv) for 36 h. The excess reagents were removed by
filtration, and the beads were washed with CH2Cl2 (50 mL) and
DMF (50 mL). Next, the resin was incubated with 1,5-diamino-
pentane (20 equiv) in 15 mL of CH2Cl2 to furnish the Dde linker.
Coupling of the first amino acid and the rest of peptide synthesis
followed standard Fmoc/HBTU chemistry using 4 equiv of Fmoc
amino acids. To construct the random region, the resin was split
into the desired number of equal portions and each portion was
4-[4-(Dimethylamino)phenylazo]benzoic Acid Hydrazide.
To a solution of Dabcyl-OSu (0.366 g, 1 mmol) in DMF was
added dropwise anhydrous NH2NH2 (31.4 μL, 1.0 mmol)
at room temperature. The mixture was stirred for 20 min. The
solvent was removed under vacuum, and the solid residue was
washed with H2O and dried under vacuum to give 0.253 g
(89.4%) of the product as a red solid: 1H NMR (400 Hz,
DMSO-d6) δ 9.86 (s, 1H), 7.97-7.93 (m, 2H), 7.82-7.77 (m,
4H), 6.86-6.82 (m, 2H), 4.55 (brs, 2H), 3.08 (s, 6H); 13C NMR
(100 Hz, DMSO-d6) δ 165.3, 154.0, 152.9, 142.7, 133.6, 128.1,
125.1, 121.6, 111.6, 39.9; HRMS (ESI) calcd for C15H17N5ONa
(Mþ þ Na) 306.1331, found 306.1323.
MetAP Library Screening (method A). A typical screening
involved 20 mg of sublibrary III (∼71000 beads), which was
transferred into a disposable Biospin column (2.0 mL). The resin
was washed with DMF (5 ꢀ 1.8 mL), H2O (5 ꢀ 1.8 mL), and a
MetAP screening buffer [30 mM Hepes (pH 7.4), 150 mM NaCl,
and 0.1 mM CoCl2]. After incubation with the screening buffer for
10 min, the resin was treated with 75-500 nM MetAP at room tem-
perature for 30-180 min. The reaction was terminated via removal
of the enzyme solution (via filtration) and the resin washed with a
0.2 M EDTA solution (5 ꢀ 1.8 mL). After a 10 min incubation with
0.2 M EDTA, the resin was washed with 1 M NaCl (5 ꢀ 1.8 mL)
and incubated with 1 M NaCl for 10 min. The resin was next
washed with H2O (5 ꢀ 1.8 mL) and DMF (5 ꢀ 1.8 mL) and treated
with 1.2 equiv of a 39:1 (mol/mol) Fmoc-OSu/Dabcyl-β-Ala-OSu
mixture dissolved in a 9:1 (v/v) DMF/phosphate buffer (50 mM,

5590 Biochemistry, Vol. 49, No. 26, 2010
Xiao et al.
pH 8.0) for 1.5 h. The resulting resin was washed with DMF (5 ꢀ
1.8 mL) and incubated with 20% piperidine in DMF for 5 and
15 min. The resin was washed with DMF (5 ꢀ 1.8 mL) and H2O
(5 ꢀ 1.8 mL) and incubated overnight with 100 mg/mL CNBr in
70% formic acid at room temperature. After the overnight
incubation, the resin was thoroughly washed with 70% HCOOH
and transferred into a 60 mm ꢀ 15 mm Petri dish with H2O. After
the addition of a drop of 6 M HCl into the Petri dish, the red-
colored beads were manually removed from the dish using a
micropipet with the aid of a dissecting microscope. A control
reaction without MetAP produced no colored beads.
MetAP Library Screening (method B). Sublibrary I or II
(typically 4.3 mg) was washed and treated with MetAP as
described above. After the MetAP reaction, the resin was washed
exhaustively with H2O and then treated with 2 equiv of NaIO4
dissolved in 0.01 M sodium phosphate buffer (pH 7.0) for 5 min at
room temperature. The reaction solution was removed, and the
resin was washed with H2O. The resin was suspended in 0.9 mL of
1% ethylene glycol in H2O and incubated for 10 min. The resin was
washed with H2O (5 ꢀ 0.9 mL) and treated with 0.1 equiv of
Dabcyl-NHNH2 in a 7:3 (v/v) mixture of 30 mM NaOAc buffer
(30 mM, pH 4.5) and MeCN for 1.5 h at room temperature. The
reaction solution was removed, and the resin was washed with
MeCN (5 ꢀ 0.9 mL) and DMF (5 ꢀ 0.9 mL). The resin was treated
with 10 equiv of NaBH3CN in 0.9 mL of DMF containing 1%
HOAc for 36 h. After that, the resin was washed with DMF (5 ꢀ
0.9 mL) and H2O (5 ꢀ 0.9 mL) and transferred into a Petri dish
with H2O. The solution was acidified with HCl, and the red-colored
beads were manually removed from the library.
Peptide Sequencing by Partial Edman Degradation
Mass Spectrometry (PED-MS). Positive beads derived from
the same screening experiment were pooled into a single reaction
vessel, suspended in 160 μL of a 2:1 (v/v) pyridine/water mixture
containing 0.1% triethylamine, and mixed with an equal volume of
an 80:1 (mol/mol) PITC/Fmoc-OSu mixture in pyridine (625 mM
PITC and 7.8 mM Fmoc-OSu). The reaction was allowed to
proceed for 6 min at room temperature, and the beads were washed
sequentially with pyridine, CH2Cl2, and TFA. The beads were
treated twice with 500 μL of TFA for 6 min each. The beads were
washed with CH2Cl2 and pyridine, and the PED cycle was repeated
five (for hits from method A) or six times (for hits from method B).
Finally, the beads were treated twice with 1 mL of 20% piperidine
in DMF at room temperature (5 min each). The beads were washed
exhaustively with water and transferred into individual microcen-
trifuge tubes (one bead/tube). Each bead was treated with 10 μL of
1.5% hydrazine in a THF/H2O mixture (1:1) at room temperature
for 15 min. The solution was acidified by the addition of 10 μL of
7% TFA in H2O. The solvent was removed under vacuum, and the
released sample was dissolved in 5 μL of 0.1% TFA in water. One
microliter of the peptide solution was mixed with 2 μL of saturated
R-cyano-4-hydroxycinnamic acid in acetonitrile and 0.1% TFA
(1:1), and 1 μL of the resulting mixture was spotted onto a 384-well
sample plate. Mass spectrometry was performed at the Campus
Chemical Instrument Center of The Ohio State University on a
Bruker III MALDI-TOF instrument in an automated manner.
The data obtained were analyzed by Moverz software (Proteo-
metrics LLC, Winnipeg, MB).
deprotection with a modified reagent K (7.5% phenol, 5% water,
5% thioanisole, 2.5% ethanedithiol, and 1% anisole in TFA), the
crude peptides were precipitated in cold diethyl ether and purified
˚
by reversed-phase HPLC on a C18 column (Varian, 120 A, 100
mm ꢀ 250 mm). The identity of the peptides was confirmed by
MALDI-TOF mass spectrometric analyses.
MetAP Activity Assay. A typical assay reaction mixture
(total volume of 100 μL) contained 1ꢀ MetAP reaction buffer
[30 mM Hepes (pH 7.4), 150 mM NaCl, and 0.1 mM CoCl2] and
0-200 μM peptide substrate. The reaction was initiated by
the addition of MetAP (final concentration of 5-1000 nM) and
quenched after 10-90 min with the addition of three drops of TFA.
The reaction mixture was centrifuged at 15000 rpm in a micro-
centrifuge for 5 min, and the clear supernatant was analyzed by
reversed-phase HPLC on an analytical C18 column (Vydac C18,
˚
300 A) eluted with a linear gradient of acetonitrile in water contain-
ing 0.05% TFA (from 10 to 40% acetonitrile over 18 min). The per-
centage of substrate-to-product conversion was determined from
the peak areas of the remaining substrate and the reaction product
(monitored at 214 nm) and kept at <20%. The initial rates were
calculated from the conversion percentages and plotted versus [S].
Data fitting against the Michaelis-Menten equation V=Vmax[S]/
(KM þ [S]) or the simplified equation V = kcat[E][S]/KM (when
KM . [S]) gave the kinetic constants kcat, KM, and/or kcat/KM.
DNA Constructs and Cell Transfection. The coding
sequences of BTF3L4, phosphoglucomutase (PGM1), and thior-
edoxin-like protein 1 (TXNL1) proteins were amplified by the
polymerase chain reaction (PCR) from the cDNA pool of HE-
K293T cells with the introduction of EcoRI and BamHI restriction
sites. The DNA primers used for PCR amplification of the BTF3L4
gene were 50-CGCGAATTCAGCATGAATCAAGAAAAGT-
TAGCC-30 and 50-CCGGGATCCGTTAGCTTCATTCTTTG-
ATGC-30. The primers used for the PGM1 gene were 50-CGCG-
AATTCGCGATGGTGAAGATCGTGACAGTTAAG-30 and
50-CGCGGATCCGGTGATGACAGTGGGTGC-30.Theprimers
used for the TXNL1 gene were 50-GAGCGGCCGCCATGGT-
GGGGGTGAAG-30 and 50-CGCGCGGATCCGTGGCTTTC-
TCCTTTTTTGC-30. The PCR products were cloned into the
p3xFLAG-CMV-14 vector (Sigma), resulting in the addition of a
C-terminal 3xFlag tag. The final constructs were verified by DNA
sequencing. The plasmids for transfection were isolated from E. coli
using the PureLink HiPure Plasmid Filter Purification Kit and
PureLink HiPure Precipitator Module (Invitrogen).
HEK293T cells were grown in a humidified environment at
37 °C with 5% CO2 in low-glucose DMEM (Gibco) supplemen-
ted with 10% FBS (Gibco) and 1% penicillin/streptomycin
(Gibco) unless otherwise stated. One day prior to transfection,
the cells were seeded at a density of 2 ꢀ 106 cells per 10 cm Petri
dish containing 10 mL of medium. Immediately before transfec-
tion, 10 μg of the appropriate plasmid DNA (p3xFLAG-CMV-
14-BTF3L4, p3xFLAG-CMV-14-PGM1, or p3xFLAG-CMV-
14-TXNL1) was added to 1 mL of serum-free DMEM, to which
25 μL of Superfect transfection reagent (Qiagen, 301305) was
added. The mixture was incubated at room temperature for
10 min. The cell medium was then changed, and the transfection
mixture was added in a dropwise fashion.
Initiator Methionine Release Assay. This assay was a
modification of a previously described procedure (28, 29). At
19.5 h post-transfection, the cells were treated for 4.5 h with either
vehicle (DMSO), MetAP1 inhibitor IV-43 (30) (10 μM), MetAP2
inhibitor TNP-470 (100 nM), or both in 4 mL of methionine-free
DMEM supplemented with 4.5 g/L glucose and sodium pyruvate
Synthesis of Individual Peptides. Individual peptides were
synthesized using standard solid-phase Fmoc/HBTU chemistry.
Each peptide was synthesized on 100 mg of CLEAR-amide resin
(0.49 mmol/g). The ninhydrin test was used to monitor the
completion after each coupling reaction. After cleavage and

Article
Biochemistry, Vol. 49, No. 26, 2010 5591
Scheme 1: Synthesis of the MetAP Substrate Librarya
aReagents and conditions: (a) glutaric anhydride (3 equiv), DIPEA, CH2Cl2; (b) 5,5-dimethylcyclohexane-1,3-dione (4 equiv), EDC
(2 equiv), DMAP (2 equiv), CH2Cl2; (c) NH2(CH2)5NH2 (20 equiv), CH2Cl2; (d) solid-phase peptide synthesis using Fmoc chemistry,
split-and-pool synthesis at random positions, and deprotection; (e) 1.5% NH2NH2 in a 1:1 THF/H2O mixture.
(Mediatech) and 10% dialyzed FBS (Gibco). For treatment for the
final 4 h, the cells were incubated with 0.2 mCi of [35S]methionine
(PerkinElmer, NEG709A). The medium was removed by aspira-
tion, and the cells were resuspended in 5 mL of ice-cold PBS, collec-
ted by centrifugation at 225g, and resuspended using a Pasteur pipet
in 1 mL of ice-cold RIPA buffer [50 mM Tris-HCl, 150 mM NaCl,
1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS (pH 8.0)]
containing 1ꢀ protease inhibitor cocktail (Roche, 11872580001).
Following a 10 min incubation (at 4 °C), the cell lysate was
centrifuged at ∼16000g for 20 min (4 °C), and the cleared super-
natant was transferred to a fresh tube containing 30 μL of anti-Flag-
conjugated agarose resin (Sigma, A2220) that had been equilibrated
by being washed with RIPA buffer (3 ꢀ 1 mL). After incubation for
1.5 h on a rotating wheel (4 °C), the resin was collected by
centrifugation at 500g for 30 s (4 °C) and washed five times with
0.9 mL of ice-cold RIPA buffer (by inverting the tube 15 times
between centrifugation steps). Finally, the resin was washed twice
with 0.9 mL of ice-cold MetAP reaction buffer [50 mM Hepes,
150 mM NaCl, and 100 μM CoCl2 (pH 7.5)] before being resus-
pended in 250 μL of MetAP reaction buffer and split into two equal
aliquots. Each aliquot was centrifuged at 500g, and the supernatant
was aspirated. The supernatant from one aliquot was discarded (the
“no enzyme” tube), and 50 μL of MetAP reaction buffer was added
to the resin. The supernatant from the other aliquot (the “enzyme
reaction tube”) was set aside as the “wash” sample, and recombi-
nant MetAP1 and MetAP2 (5 μM each) were added to the resin
suspended in 50 μL of MetAP reaction buffer. After incubation
overnight (∼16 h) at room temperature, 75 μL of MetAP reaction
buffer was added to each tube; then the resin was pelleted, and the
supernatant was transferred to a fresh tube. The 35S content of each
sample (including the wash sample) was determined when each
sample was mixed with 1 mL of scintillation fluid (PerkinElmer,
1200-439) and scintillation counting on a 1450 MicroBeta appara-
tus (Wallac). The percentage of [35S]methionine release was calcu-
lated from the ratio of counts in the reaction buffer to the sum of
these counts and the counts from the corresponding resin sample.
the form of NH2-MX1X2X3X4X5LNBBR-Dde-resin [where B is
β-alanine and X1-X5 are 2-aminobutyrate (Abu or U) and
norleucine (Nle or M), which were used as Cys and Met surrogates,
respectively, or any of 16 proteinogenic amino acids except for Arg,
Cys, Lys, and Met] was synthesized on PEGA resin (0.4 mmol/g,
150-300 μm in diameter when swollen in water) (Scheme 1). A
methionine was placed at the N-termini of all sequences because it is
already well established that all MetAPs have a stringent require-
ment for Met as the P1 residue (21, 31). Arg, Lys, Cys, and Met
were excluded (or replaced) from the random region because they
would interfere with the screening procedure (vide infra). The
invariant sequence LNBBR was added to facilitate MALDI MS
analysis, by providing a fixed positive charge (Arg) and shifting
the mass of all peptides to >500 (to avoid overlap with MALDI
matrix signals). The peptides were covalently linked to the PEGA
resin via a Dde linker, which permits facile peptide release by
treatment with hydrazine prior to MS analysis. To construct the
Dde linker, the free amino group on PEGA resin was first acylated
with glutaric anhydride, and the resulting carboxylic acid (1)
was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) and condensed with 5,5-dimethylcyclohexane-1,3-dione to
give enol 2 (32). Subsequent reaction of enol 2 with excess 1,5-
diaminopentane provided the desired Dde linker 3 containing a
free amine as the anchor for peptide synthesis. After the addition
of the LNBBR sequence using standard Fmoc/HBTU chemistry,
the random region was synthesized via the split-and-pool
method (33, 34). After the addition of the last random residue
(X1), the library was split into three sublibraries. Sublibraries I
and II contained Ser and Thr as the X1 residue, respectively,
whereas in sublibrary III, the X1 residue represented an equimo-
lar mixture of the other 16 amino acids. The theoretical diversity
of the library is 185 or 1.9 ꢀ 106.
MetAP Library Screening. Two different methods were
devised to screen the MetAP substrate library. Method A (Scheme
2a) was designed for sublibrary III, which was subjected to limited
MetAP reaction, so that only those beads that carried the most
efficient substrates of MetAP would undergo partial cleavage of the
N-terminal methionine (usually a few percent), while the vast
majority of beads (which displayed poorer substrates or inactive
peptides) would have little or no reaction. The library was then
RESULTS AND DISCUSSION
Design and Synthesis of the MetAP Substrate Library.
A
one-bead-one-compound (OBOC) MetAP substrate library in

5592 Biochemistry, Vol. 49, No. 26, 2010
Xiao et al.
Scheme 2: Two Methods for Library Screening against MetAP
treated with a 39:1 (mol/mol) mixture of Fmoc-OSu and Dabcyl-
β-Ala-OSu, a red dye molecule. This resulted in the N-terminal
acylation (or carbamoylation) of all library peptides (both MetAP
reaction products and unreacted substrates), and all of the library
beads became red (due to attachment of the Dabcyl moiety). Next,
the library was treated with piperidine to remove the N-terminal
Fmoc group, followed by CNBr to cleave any N-terminal methio-
nine. For beads with peptides that did not undergo any MetAP
reaction, CNBr would remove the N-terminal methionine, along
with the Dabcyl group, rendering these beads colorless. On the
other hand, any MetAP reaction products would not be affected
by the CNBr treatment because they no longer contained any
methionine in their sequences. Thus, those beads that had under-
gone significant MetAP reaction (positive beads) would retain the
Dabcyl group and remain red. The positive beads were manually
removed from the library using a micropipet, and their peptide
sequences were determined by PED-MS (27). It is worth noting
that the use of a 39:1 Fmoc-OSu/Dabcyl-β-Ala-OSu mixture was
critical for successful library screening, as it ensured that there were
sufficient amounts of peptides with free N-termini (after Fmoc
removal) for PED-MS analysis. It also improved the color con-
trast between positive and negative beads. When only Dabcyl-
β-Ala-OSu was used for N-terminal acylation, all of the library
beads retained significant red color after the CNBr treatment,
because methionine removal by CNBr was not quantitative.
The method described above was less effective for sublibraries I
and II (which contained Ser and Thr at the P10 position, respec-
tively) because CNBr cleavage of methionine is inefficient when
Ser or Thr is next to methionine (35). The β-hydroxyl group of Ser
(or Thr) can intramolecularly react with the five-membered ring
intermediate, resulting in the conversion of the methionine into a
homoserine instead of peptide bond cleavage. We therefore devel-
oped an alternative approach (method B) for those two sublibraries
(Scheme 2b). Enzymatic cleavage of the N-terminal methionine
from the library peptides generated an N-terminal Ser or Thr,
which was selectively oxidized by sodium periodate into a glyoxyl
group. The newly formed aldehyde group was then selectively
conjugated with Dabcyl-hydrazide and reduced with NaBH3CN.
Again, the resulting positive (red-colored) beads were readily iden-
tified from the library and sequenced via PED-MS.
Substrate Specificity of E. coli MetAP1. A total of 105 mg
of sublibrary III (∼3.9 ꢀ 105 beads) was screened against E. coli
MetAP1 in five separate experiments (25 mg of resin each). The
most colored beads were isolated and sequenced by PED-MS to
give 236 complete sequences, which should represent the most
efficient substrates of E. coli MetAP1 (Table 1). Similar sequences
were selected from each screening experiment, demonstrating the
reproducibility of the screening method. E. coli MetAP1 has the
most stringent specificity at the P10 position, with Ala being the
most preferred amino acid (in 84% of all selected sequences),
followed by glycine (10%) and 2-aminobutyrate (6%) (Figure 1a).
At the P20 position, E. coli MetAP1 tolerates a wide variety of
amino acids, but with preference for hydrophilic residues (Glu, Asp,
Asn, Gln, Ser, and Thr) or small residues such as Ala and Abu. A
few of the selected peptides also contained a Phe at the P20 position.
Interestingly, of the 236 selected peptides, none contained a proline,
histidine, or tryptophan at this position. The enzyme also has broad
specificity at the P30 position, where hydrophobic residues were
frequently selected, whereas acidic residues (especially Asp), Gly,
His, and Trp were underrepresented. There appears to be a negative
correlation between the P20 and P30 positions with respect to acidic
amino acids. When the P20 residue was acidic, the P30 residue was
almost always hydrophobic, and vice versa, when Asp or Glu was
present at the P30 position, the P20 residue was typically hydro-
phobic (Table 1). None of the most preferred substrates contained
acidic residues at both P20 and P30 positions. No obvious selectivity
was observed at the P40 or P50 position. Screening of sublibraries I
and II (9 mg each) by method B revealed a similar specificity profile
at the P30-P50 positions, although Ala and acidic residues (Asp
and Glu) were more frequently selected at the P20 position when
Ser or Thr was the P10 residue (Figure 1b and Table S1 of the
Supporting Information).

Article
Biochemistry, Vol. 49, No. 26, 2010 5593
Table 1: Most Preferred Substrates of E. coli MetAP1 Selected from
Sublibrary III (236 total)a
AAADV
AADEI
AAELI
AEUEI
ANVIN
ANVHI
ANVIG
ANYGA
ANYQG
AQAGE
AQFAG
AQFGP
AQGUI
AQLVG
AQNVU
AQNIP
AQNFT
AQNFP
AQQUH
AQQUD
AQSAN
AQSMI
AQSMI
AQSVQ
AQSNI
AQSTY
AQSSA
AQSHU
AQTEM
AQTAS
AQVUT
AQVGI
ASAES
ASFUU
ASINE
AUPSE
AUSFG
AUTAN
AUTNP
AUUNH
AUUFG
AUUEF
AUVHT
AVEGY
AVGFP
AVNHI
AVTHT
AVTEU
AVUAD
AVUDS
AYNQQ
AYNNU
AYSET
AYSSG
AYVSL
GAEIE
AEUEG
AEVMH
AEVMA
AEVMD
AEVNI
AEVTE
AEVIS
AAFNT
AAFTQ
AAIST
AALHQ
AAMEP
AANDI
AANN I
AAPEI
AEVDI
AEYPV
AEYGA
AFAEG
AFDND
AFEGP
AFNUG
AFNQS
AFPGI
AATSI
AATYP
AATNT
AAUIS
AAUHI
AAUMD
AAASU
ADAAL
ADAFE
ADFEU
ADLMP
ADMDI
ADPEM
ADSLG
ADSAF
ADTQF
ADTIN
ADTAI
ADUGA
ADUAA
ADWDM
ADYNI
AEAFD
AEANY
AEFEM
AEFHE
AEISI
AFPNT
AFPSP
AFSHS
AFSTH
AFSDI
GAMHE
GAUEY
GDFDF
GDMLP
GDVSL
GEUID
GFADQ
GFAGE
GFIDN
GFSEI
AFSNS
AFSQT
AFTMP
AFVGP
AGGPD
AIDED
ALAGE
ALUVU
AMAEQ
AMANI
AMEVD
AMNSG
AMUAP
ANADF
ANAEQ
ANAEH
ANFUT
ANISI
ASLEV
ASLNS
ASMHQ
ASNVG
ASNIT
GFSQE
GFSSQ
GLAHG
GLTDT
GMNEP
GMUEE
GMUTE
GNFEE
GNLME
GSLES
ASSMN
ASTHE
ASUTL
ASVSM
ASVHV
ASVVS
ASYSU
ATFTE
ATIEE
AEIED
AEIEIb
AEIIG
ANIDE
ANLNV
ANLEQ
ANMIG
ANMTA
ANMDF
ANNHE
ANSQY
ANTQL
ANTVG
ANTEA
ANTYT
ANTVA
ANUSI
ANUID
ANUEM
ANUQY
ANVED
ANVIY
AELID
AELFA
AELEE
AELVU
AEMEI
AEMSN
AEMNQ
AEMEW
AENYP
AEPIV
GSTLE
GSUEF
GUEEF
UDYGD
UEFEE
UEMFE
UETIN
UFSEV
UFUET
UNQIH
UNVHN
UNVDM
UQGEF
UQUNG
USMED
USUIT
ATMNS
ATQPI
ATUYT
ATVAU
AUADF
AUEYD
AUFGA
AUGEF
AUIPS
AESTF
AETLH
AETTY
AETID
AEUVG
AEUGG
AEUEH
AEUQF
F
IGURE 1: Substrate specificity of E. coli MetAP1. (a) Most preferred
substrates selected from sublibrary III (total of 236 sequences). (b) Most
preferred substrates from sublibraries I and II (total of 124 sequences).
(c) Val-containing sequences (at position P10) derived from medium-
colored beads in sublibrary III (total of 92 sequences). (d) Ala-, Abu-,
and Gly-containing sequences (at position P10) derived from medium-
colored beads in sublibrary III (total of 140 sequences). (e) Sequences
derived from colorless beads in sublibrary III (total of 169 sequences).
Displayed are the amino acids identified at each position (P10-P50).
Occurrence on the y-axis represents the number of selected sequences
that contained a particular amino acid at a certain position.
AUIGP
AUIAP
AUPAI
AUPSN
AUPSE
UTDLE
UUEID
aSequences were obtained from five screening experiments performed at
75-100 nM E. coli MetAP1. U, R-
-aminobutyric acid; M, norleucine. bPeptide
selected for further kinetic analysis.
L
To identify the less efficient substrates and MetAP-resistant
sequences, sublibrary III (40 mg) was treated exhaustively with a
high concentration of E. coli MetAP1 (1.0 μM) for an extended
period of time (overnight). In addition to intensely colored beads,
this screening experiment also produced many medium-colored
and lightly colored beads (∼15% of all library beads), which
should represent the less efficient substrates of E. coli MetAP1.
We randomly selected some of the medium-colored beads for
sequence analysis and obtained 232 complete sequences (Table
S2 of the Supporting Information). Our results showed that 40%

5594 Biochemistry, Vol. 49, No. 26, 2010
Xiao et al.
of these less efficient substrates contained a Val as the P10 residue,
while the rest of peptides had Gly (27%), Abu (23%), and Ala
(10%) at this position. When the subset of sequences that contained
Val as the P10 residue was analyzed, the specificity profile at the
P20-P50 positions was essentially identical to that observed for the
most efficient substrates (Figure 1c). Similar analysis of the other
subset of sequences (which contained Gly, Abu, or Ala as the P10
residue) showed that they usually contained less favorable amino
acids at the P20 (e.g., Gly, Ile, Leu, and Pro) and/or P30 positions
(e.g., Asp, Glu, and His) (Figure 1d). Sequence analysis of 171
randomly selected colorless beads from the library showed that
most of the MetAP-resistant peptides contained amino acids with
large side chains like the P10 residue, while none of them had Ala at
the P10 position (Figure 1e and Table S3 of the Supporting Infor-
mation). Only six of the peptides contained Gly (MGDVFQ and
MGPTET), Abu (MUHINS, MUPETF, and MUILVE), or Val
(MVLFHH) at the P10 position. These peptides either contained a
disfavored residue at the P20 position (Pro or His) or were very hy-
drophobic (MUILVE and MVLFHH), making the beads poorly
swelled in aqueous solution and thus poorly accessible to the
enzyme during library screening. As expected from the large num-
ber of colorless beads (∼75% of all beads), there was no obvious
“selectivity” at the P20-P50 positions. These data indicate that all
N-methionyl peptides containing a small P10 residue (Ala, Gly,
Abu, Ser, Thr, and Val) are accepted as E. coli MetAP1 substrates,
albeit with different catalytic efficiencies.
Substrate Specificity of Human MetAP1. Human Me-
tAP1 was similarly screened against sublibraries I-III to identify
peptide sequences that are most preferred or less preferred by the
enzyme, and resistant to the enzyme. The most intensely colored
beads were selected from sublibraries III (60 mg) and I (9 mg) and
sequenced to give 128 and 74 unambiguous sequences, respec-
tively (Tables S4 and S5 of the Supporting Information). Similar
sequences were selected from the two sublibraries (Figure 2a,b).
In general, human MetAP1 has a specificity profile similar to that
of E. coli MetAP1 but also has some unique features. Like the
E. coli enzyme, human MetAP1 prefers Ala at the P10 position,
but this preference is stronger than that of E. coli MetAP1 (124 of
the 128 best substrates had Ala as the P10 residue). Only four
peptides contained Gly (three peptides) or Abu (one peptide) as
the P10 residue. The underrepresentation of Abu at this position
suggests that amino acids with larger (than a methyl group) side
chains are poorly tolerated by the human MetAP1 active site.
This notion is further supported by the data from sublibrary II
(which contained Thr as the P10 residue) (Figure 2c and Table S6
of the Supporting Information). Screening of sublibrary II
against human MetAP1 produced only lightly colored beads,
despite the use of a higher enzyme concentration (800 nM) and a
longer reaction time (2 h), suggesting that any peptide containing
a Thr at the P10 position is a relatively poor substrate of human
MetAP1. Other features shared with the E. coli enzyme include
the absence (or underrepresentation) of Pro at the P20 position
and His and Trp at both P20 and P30 positions among the most
active substrates. The most striking difference between the E. coli
and human MetAP1 enzymes lies in their different tolerance to
acidic residues. While E. coli MetAP1 prefers Asp and Glu at P20,
P40, and P50 positions (Figure 1), the human enzyme strongly
disfavors acidic residues at all positions. Of the 245 most pre-
ferred substrates selected from sublibraries I-III, only one
sequence [MA(Nle)DWE] contained acidic residues. Another
difference is that while E. coli MetAP1 disfavors Gly at positions
P20 and P30, the human enzyme has no such discrimination.
F
IGURE 2: Substrate specificity of human MetAP1. (a) Most preferred
substrates selected from sublibrary III (total of 128 sequences). (b) Most
preferred substrates from sublibrary I (total of 74 sequences). (c) Most
preferred substrates selected from sublibrary II (total of 43 sequences).
(d) Ala-, Abu-, and Gly-containing sequences (at position P10) derived
from medium-colored beads in sublibrary III (total of 50 sequences).
(e) Val-containing sequences (at position P10) derived from medium-
colored beads in sublibrary III (total of 24 sequences). Displayed are the
amino acids identified at each position (P10-P50). Occurrence on the
y-axis represents the number of selected sequences that contained a
particular amino acid at a certain position.
The less optimal substrates and inactive peptides were identified
by treating sublibrary III (12 mg) with a high concentration of
human MetAP1 (2.7 μM) for 24 h and randomly selecting a frac-
tion of the medium-colored and colorless beads for sequence ana-
lysis. The less optimal substrates contained Val (33%), Gly (28%),

Article
Biochemistry, Vol. 49, No. 26, 2010 5595
Abu (24%), and Ala (15%) as the P10 residue (Table S7 of the
Supporting Information). Of the 48 Gly-, Abu-, and Ala-contain-
ing (at position P10) peptides, 35 contained at least one acidic
residue, often at the P20 and/or P30 position, while three other
peptides had Pro as the P20 residue (Figure 2d). The Val-containing
peptides (at position P10) contained no Pro at the P20 position and
many fewer acidic residues in their sequences (Figure 2e). None of
the MetAP1-resistant peptides contained Ala or Gly at the P10
position, while two peptides had Abu or Val as the P10 residue
(Figure S1 of the Supporting Information). These data pro-
vide further support for our conclusion that a larger P10 residue
(e.g., Val and Thr), a Pro at the P20 position, and/or acidic
residues at positions P20-P50 can decrease the catalytic activity
toward human MetAP1. They also suggest that human MetAP1,
like the E. coli enzyme, will accept essentially all N-methionyl
peptides containing Ala, Gly, Abu, Ser, Thr, or Val at the P10
position as substrates, although the Thr- and Val-containing
peptides are generally inefficient substrates.
Substrate Specificity of Human MetAP2. Sublibraries I
(4.3 mg), II (4.3 mg), and III (80 mg) were screened against
human MetAP2, and the most intensely colored beads were
sequenced to give 96, 65, and 632 complete sequences, respectively
(Tables S8-S10 of the Supporting Information). In addition, 88
medium-colored beads were isolated from sublibrary III and
sequenced (Table S11 of the Supporting Information). In general,
human MetAP2 has a specificity profile very similar to that of
human MetAP1 (Figure 3). For example, both human MetAP1
and MetAP2 disfavor Pro at position P20, Trp at positions P20 and
P30, and acidic residues at positions P20-P50. The main difference
between the two enzymes is their specificity at the P10 position.
While only one of the 128 most preferred substrates of human
MetAP1 contained an Abu as the P10 residue, MetAP2 selected
Abu with the second highest frequency (122 of 632 sequences),
more frequently than Gly (90 sequences). Moreover, 19 of the most
preferred substrates had a Val as the P10 residue. This suggests that
the S10 site of human MetAP2 is more capable of accommodating
amino acids with larger side chains than MetAP1 does. This notion
is also supported by our data on sublibrary II. We found that
treatment of sublibrary II with 250 nM human MetAP2 generated
intensely colored beads in 2 h, while treatment with 800 nM human
MetAP1 for 2 h produced only lightly colored beads. We noted that
unlike E. coli and human MetAP1, MetAP2 selected a significant
number of His residues at positions P20 and P30. The underlying
reason is not yet clear.
Kinetic Properties of Selected MetAP Substrates. Fifteen
peptides were synthesized individually on a larger scale, purified by
HPLC, and assayed against the three MetAPs to confirm the
screening results as well as provide specificity data on amino acid
residues excluded from the library (i.e., Arg and Lys) (Table 2,
entries 1-15). Peptides 1 and 2 are two of the most preferred
substrates of E. coli MetAP1, selected from sublibraries III and I,
respectively (Table 1 and Table S1 of the Supporting Information).
Peptide 12 is a preferred substrate of human MetAP2 selected from
sublibrary III (Table S8 of the Supporting Information). The rest of
the peptides are variants of peptides 1 and 12, designed to test the
effect of the P10-P30 residues on MetAP activity. A Tyr was added
to the C-terminus of each peptide to facilitate their concentration
determination. Since most of the peptides did not reach saturation
at the highest concentration tested (1.0 mM), only their kcat/KM
values are used for comparison.
F
IGURE 3: Substrate specificity of human MetAP2. (a) Most pre-
ferred substrates selected from sublibrary III (total of 632 sequences).
(b) Most preferred substrates from sublibraries I and II (total of 144
sequences). (c) Ala-, Abu-, and Gly-containing sequences (at position
P10) derived from medium-colored beads in sublibrary III (total of 52
sequences). (d) Val-containing sequences (at position P10) derived
from medium-colored beads in sublibrary III (total of 16 sequences).
Displayed are the amino acids identified at each position (P10-P50).
Occurrence on the y-axis represents the number of selected sequences
that contained a particular amino acid at a certain position.
respectively (Table 2). Replacement of the Ala at the P10 position
with Gly, Pro, Thr, and Val decreased the activity by 3.4-, 4.7-,
9.3-, and 44-fold, respectively. This is largely consistent with the
screening results, except for the results for the proline-containing
peptide. Previous studies have also shown that peptides con-
taining Pro as the P10 residue are efficiently hydrolyzed by
MetAPs (17-25). Thus, the absence of Pro from the P10 position
among the selected substrates was likely caused by the bias of the
screening procedure (method A). Proline, which bears a second-
ary amine, is significantly less reactive with respect to activated
esters such as Dabcyl-β-Ala-OSu than the other 19 proteinogenic
amino acids (36). Presumably, MetAP reaction products that
contained an N-terminal proline were not efficiently labeled with
Dabcyl-β-Ala-OSu and became false negatives. Replacement of
As expected, peptides 1 and 2 were highly active toward the E.
coli enzyme, having kcat/KM values of 14000 and 28000 M-1 s-1
,

5596 Biochemistry, Vol. 49, No. 26, 2010
Xiao et al.
Table 2: Kinetic Constants of E. coli and Human MetAPs against Selected Peptidesa
kcat/KM (M-1 s-1
)
entry
peptide
E. coli MetAP1
human MetAP1
human MetAP2
relative activity of MetAP2/1
1
MAEIEIYb
MSDFIGYb
MGEIEIY
MPEIEIY
14000 ( 3900
28000 ( 1100
4100 ( 120
3000 ( 1490
1500 ( 50
380 ( 110
18900 ( 1300
13500 ( 7600
NA
1700 ( 160
ND
1040 ( 50
ND
0.63
ND
3.6
2
3
180 ( 8
160 ( 8
29 ( 2
660 ( 20
140 ( 60
460 ( 60
450 ( 180
23300 ( 1700
ND
4
0.88
16
5
MTEIEIY
6
MVEIEIY
NA
>200
3.5
7
MARIEIY
MAGIEIY
MAPIEIY
6600 ( 600
ND
8
ND
0.43
1.4
9
1300 ( 20
3580 ( 350
∼7
540 ( 25
10
11
12
13
14
15
16
17
18
19
20
MAHIEIY
MVKIEIY
MAHAIHYb
MAGAIHY
MAWIEIY
MAEWEIY
MVHQVLYc
MVNPTVYc
MVLSPAYc
MVGVKPYc
MVAAAAYc
14500 ( 550
ND
5000 ( 3800
860 ( 210
36000 ( 1900
37000 ( 6200
4900 ( 750
2160 ( 800
4000 ( 240
2600 ( 60
2600 ( 1000
2070 ( 280
10000 ( 1000
116
0.88
3.7
ND
41000 ( 18000
10000 ( 340
9600 ( 350
5300 ( 90
64 ( 5
ND
ND
0.51
0.41
63
ND
ND
ND
13 ( 2
15 ( 1
28 ( 1
130 ( 6
200
175
74
ND
ND
ND
79
aAll peptides contained a free N-terminus and a C-terminal amide. NA, no detectable activity; ND, not determined. bPeptides selected from the
combinatorial library. cPeptides derived from human proteins.
the P20 residue (Glu) with an arginine, which was excluded from
the library for technical reasons, slightly increased the catalytic
activity (by 1.3-fold). This suggests that the frequent selection of
acidic residues at the P20 position was not due to the presence of
favorable charge-charge interactions; the E. coli enzyme simply
prefers a hydrophilic residue at this position. Indeed, other neutral,
hydrophilic residues such as Asn, Gln, and Ser were also frequently
selected at this position (Figure 1). Consistent with the absence of
Pro from the P20 position among the most preferred substrates,
peptide 9 (MAPIEIY) had an activity that was too low to be reliably
determined by the HPLC assay. A surprising finding was that
peptide 10, which contains a His at the P20 position, had excellent
activity (kcat/KM = 14500 M-1 s-1), even though His was not at all
selected at this position (Figure 1). Careful kinetic analysis showed
that at higher concentrations (>100 μM), peptide 10 inhibited
the MetAP activity (data not shown). During HPLC analysis of
the MetAP reaction mixture, we observed that peptide 10 formed a
complex with the Co2þ ion (used in MetAP assays). The pepti-
de-metal complex had a retention time different from that of
the free peptide and disappeared when a lower concentration of
Co2þ ion was used in the assay buffer. Presumably, during library
screening, the His-containing peptides inhibited the MetAP en-
zymes by binding to their active-site metal ion(s). Inhibition of
MetAP by Met-X-His peptides had also been reported by other
investigators (16).
Kinetic assays of human MetAP1 and MetAP2 against the above
peptides also confirmed their specificity profiles revealed by library
screening. Indeed, both human enzymes disfavor acidic residues.
Replacement of a Glu at the P20 position with either Arg or His
increased the catalytic activity by 2-22-fold (in Table 2, compare
peptides 1, 7, and 10). Likewise, a Glu at the P40 position reduced
the activity of both enzymes by 7-12-fold (compare peptides 10
and 12). Human MetAP2 is more tolerant than MetAP1 to larger
P10 residues. While all of the peptides containing Ala, Gly, or Pro at
the P10 position had similar activities toward the two enzymes
(MetAP2:MetAP1 ratio between 0.43 and 3.7), peptides 6 and 11,
which contain a Val at the P10 position, were 2 orders of magnitude
more active toward MetAP2 than MetAP1. MetAP2 also had a
16-fold higher activity than MetAP1 toward a Thr-containing pep-
tide (peptide 5). To test whether this is a general property of MetAP2,
we synthesized five additional Met-Val peptides, derived from the
N-terminal sequences of human proteins sulfurtransferase, cyclo-
philin A, hemoglobin R chain, thioredoxin-like protein 1 (TXNL1),
and attractin, which are known to undergo N-terminal methionine
removal in vivo (Table 2, peptides 16-20) (37). All five peptides
had kcat/KM values in the range of 2000-10000 M-1 s-1 toward
MetAP2 but were only poorly active toward human MetAP1
(74-200-fold lower activity). Our data suggest that these five pro-
teins, and likely all Met-Val- and Met-Thr-containing proteins in
humans, are mainly processed by MetAP2 in vivo. Previous X-ray
crystal structural studies have shown that human MetAP1 has a
˚
narrower active-site cleft than MetAP2 (by ∼1 A), and the reduced
size restricts the access of fumagillin and related compounds to the
active site of MetAP1 (26). Similarly, the smaller active site of
MetAP1 would sterically clash with the larger side chains of Val and
Thr at the P10 position. Finally, we tested two Trp-containing
peptides (Table 2, peptides 14 and 15) to determine the effect of Trp
on MetAP activities. Surprisingly, although none of the enzymes
selected Trp among their most preferred substrates (Figures 1-3),
both peptides (which contain a Trp at the P20 and P30 positions,
respectively) were efficient substrates of human MetAP1 and
MetAP2. A possible explanation for this discrepancy may be that
beads displaying hydrophobic Trp-containing peptides swelled
poorly in the aqueous screening buffer, rendering the peptides on
these beads poorly accessible to the MetAP enzymes.
In Vivo Validation of MetAP1 and MetAP2 Substrate
Specificity. To validate the in vitro substrate specificity data, we
examined the removal of the N-terminal Met from phosphoglu-
comutase 1 (PGM1) and TXNL1 in vivo. PGM1 and TXNL1
contain N-terminal sequences of MVKIVT and MVGVKP,
respectively, and are expected to be specific substrates of Me-
tAP2. The protein BTF3L4, which has an N-terminal sequence of

Article
Biochemistry, Vol. 49, No. 26, 2010 5597
F
IGURE 4: Retention of the initiator methionine of TXNL1 caused by inhibition of MetAP2 by TNP-470. (a) Immunoblotting (IB) of
immunoprecipitated (IP) C-terminal 3xFlag-tagged BTF3L4 protein from the HEK293T cell lysate. (b) Immunoblotting of the immunopreci-
pitated C-terminal 3xFlag-tagged PGM1 protein from the HEK293T cell lysate (3W, three washes with RIPA buffer; 6W, six washes with RIPA
buffer). (c) Immunoblotting of the immunoprecipitated C-terminal 3xFlag-tagged TXNL1 protein from the HEK293T cell lysate. (d and e)
Immunoprecipitated C-terminal 3xFlag-tagged BTF3L4, PGM1, and TXNL1 were aliquoted for [35S]methionine scintillation counting after
incubation with MetAP reaction buffer (d) or after in vitro processing by both MetAP1 and MetAP2 (e). IV-43 or IV (10 μM), TNP-470 or T
(100 nM), or both were added for the last 4.5 h. The error bars are standard errors from two (BTF3L4 and PGM1 in panel d), three (TXNL1 in
panel d and BTF3L4 and PGM1 in panel e), or five (TXNL1 in panel e) independent experiments. N-Terminally methionine unprocessed
BTF3L4, PGM1, and TXNL1 have 3, 12, and 8 methionine residues per molecule, respectively. *p = 0.02; ***p = 0.0003.
MNQEKL and is not expected to undergo N-terminal Met
removal, was used as a negative control. Human embryonic kidney
epithelial cell line HEK293T overexpressing C-terminally Flag-
tagged PGM1, TXNL1, or BTF3L4 proteins was treated with IV-
43 [a specific MetAP1 inhibitor (38)] and/or TNP-470 [a specific
MetAP2 inhibitor (13)] and then pulsed with [35S]Met to label
any newly synthesized proteins. The putative substrate proteins
(and control) were immunoprecipitated from the cell lysates with
antibodies against the Flag tag. The partially purified proteins
were then treated with recombinant MetAP1 and MetAP2 in vitro
to cleave any retained N-terminal [35S]Met residue, which was
caused by inhibition of the cellular MetAPs. Treatment of the cells
expressing TXNL1 with either TNP-470 alone or TNP-470 and IV-
43 in combination, but not IV-43 alone, resulted in a 2-fold increase
in the extent of N-terminal Met retention over the DMSO control
(Figure 4). No significant increase in the extent of Met retention
was observed for cells expressing BTF3L4 or PGM1. These results
demonstrate that TXNL1 is indeed a specific substrate of MetAP2.
The result with PGM1 was unexpected and may be caused by one
or both of the following factors. Inspection of the structure of
PGM1 shows that the retained Met would be buried in the folded
protein and thus inaccessible to MetAP action (39). Alternatively,
the N-terminal Met might be N-acetylated upon forced retention in
cells and protected against MetAP2 in vitro. In this regard,
treatment of human and mouse cells with TNP-470 resulted in
the retention and acetylation of the N-terminal Met of cyclophilin
A, a well-established MetAP2-specific substrate (40).
prefer small amino acids (e.g., Ala, Gly, Ser, Pro, Cys, Thr, and
Val) as the P10 residue (17-25). Our results confirmed these earlier
findings. Several studies demonstrated the importance of residues
at positions P20 and beyond for catalytic activity (20, 21). The only
systematic study of MetAP substrate specificity was conducted by
Frottin et al. (25), who synthesized and assayed ∼120 short peptides
against E. coli MetAP1 and P. furiosus MetAP2. Their data suggest
that removal of N-terminal methionine from proteins in vivo is
dictated by the substrate specificity of MetAP(s). Our data for
E. coli MetAP1 are largely consistent with the results of Frottin et al.,
but with one key difference. Frottin et al. reported that E. coli
MetAP1 had poor activity against peptides containing acidic resi-
dues at positions P20-P40. In contrast, our library screening and
kinetic analysis of individually synthesized peptides both showed
that the E. coli enzyme prefers acidic residues at positions P20, P40,
and P50. This discrepancy is likely due to the fact that Frottin et al.
employed very short peptides containing free C-termini (primarily
tri- and tetrapeptides), which generally had poor activities toward
the E. coli enzyme (kcat/KM ∼ 102-103 M-1 s-1). As described pre-
viously, our screening data revealed a negative correlation between
the P20 and P30 positions with regard to acidic residues (i.e., the en-
zyme disfavors the presence of negative charges at both positions).
Presumably, the negatively charged C-terminus of the peptides used
in the Frottin work interfered with the binding of acidic peptides to
the enzyme active site.
Proteomics analyses of TNP-470 treated cells have identified
several mammalian proteins as MetAP2-specific substrates, includ-
ing bovine cyclophilin A (N-terminal sequence MVNPTV) (41)
and glyeraldehyde-3-phosphate dehydrogenase (MVKVGV) (41)
Corroboration with Literature Data. Previous studies have
led to the conclusion that both bacterial and eukaryotic MetAPs

5598 Biochemistry, Vol. 49, No. 26, 2010
Xiao et al.
CONCLUSION
Table 3: Prediction of N-Terminal Met Removal in E. coli and Mammalian
We have developed a novel peptide library method to system-
atically profile the sequence specificity of bacterial and human
MetAPs. The results show that the specificity of MetAPs is
largely dictated by the P10 residue; however, the sequence at
positions P20-P50 does have a significant effect on the extent of
Met removal, especially for proteins containing less optimal P10
residues (e.g., Thr and Val). Further, we show that the primary
difference between human MetAP1 and MetAP2 is their differ-
ential tolerance for Thr and Val at the P10 position, suggesting
that MetAP2 is primarily responsible for N-terminal processing
of proteins that contain N-terminal Met-Val and Met-Thr
sequences. This difference in substrate specificity between human
MetAP1 and MetAP2 may be ultimately responsible for the
unique effects of inhibitors of MetAP2 on angiogenesis. Finally,
our data have permitted us to formulate a more complete set of
rules for predicting the fate of the N-terminal Met of bacterial
and mammalian proteins.
Cytoplasm
N-terminal sequence
Met removal?
enzyme
E. coli
<M[ACGPS][∧P]a
<M[ACGPSTV]P
<M[VT][∧P]
yes
MetAP1
no
variable
no
MetAP1
<M[DEFHIKLMNQRWY]
Mammalian
<M[ACGPS]
<M[VT][∧DEP]
yes
MetAP1 and MetAP2
MetAP2
yes
<M[VT][DEP]
variable
no
MetAP2
<M[DEFHIKLMNQRWY]
a<M indicates that the Met is at the N-terminus of a protein. [ACGPS]
indicates that the P10 residue is Ala, Cys, Gly, Pro, or Ser. [∧P] indicates that
Pro is excluded from position P20.
and human thioredoxin (MVKQI) (40), SH3 binding glutamic
acid rich-like protein (MVIRV) (40), and elongation factor-2
(MVNFT) (40). In vitro kinetic assays of the N-terminal peptides
of these proteins confirmed that they all have activity 2 orders of
magnitude higher toward MetAP2 than toward MetAP1. Thus,
together with TXNL1 identified in this work, all six MetAP2-
specific substrates contain a Val as the P10 residue, consistent
with our library screening data. One known exception is human
protein 14-3-3γ, which became incompletely processed when
either normal or tumor epithelial cells were treated with siRNA
specific for MetAP2 or MetAP1-specific inhibitor IV-43 (30, 42).
The N-terminal sequence of this protein, MVDREQ, suggests
that it is a poor substrate for both MetAP1 and MetAP2 (Table 2,
entry 6). It is thus not surprising that an increase in the level of
retention of its N-terminal methionine was observed upon
inhibition of either MetAP1 or MetAP2, suggesting that both
MetAP1 and MetAP2 are necessary to completely process its
N-terminal Met.
Predicted N-Terminal Met Removal Pattern in E. coli
and Human Proteomes. On the basis of the specificity data
from our work and the literature (43), we make the following
predictions about the N-terminal Met removal (Table 3). In
E. coli, proteins containing small residues Ala, Cys, Gly, Pro, and
Ser as the P10 residue and any amino acids other than Pro at the
P20 position are expected to undergo complete Met cleavage.
When the P10 residue is any amino acid other than Ala, Cys, Gly,
Pro, Ser, Thr, or Val or if the P20 residue is Pro, the N-terminal
Met is retained. When the P10 residue is Thr or Val and the P20
residue is not Pro, N-terminal Met cleavage is variable, depend-
ing on several factors such as the actual N-terminal sequence at
positions P20-P50, the level of protein expression, and the growth
condition of the cell. In mammalian cells, proteins containing
Ala, Cys, Gly, Pro, or Ser at the P10 position generally undergo
complete N-terminal processing, catalyzed by MetAP1, MetAP2,
or both. When the P10 residue is Thr or Val, the N-terminal Met
removal is primarily catalyzed by MetAP2 and the extent of
cleavage depends on the sequence at positions P20-P50. When the
P20 residue is not Asp, Glu, or Pro, the N-terminal processing is
expected to be complete. When Asp, Glu, or Pro is the P20
residue, Met removal is either incomplete or does not occur. It
should be noted that these rules are meant to be a general guide-
line and occasional exceptions have been observed.
SUPPORTING INFORMATION AVAILABLE
Tables containing the peptide sequences selected against the
three MetAP enzymes and additional figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
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