Tài liệu Báo cáo khoa học: Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites doc

Different modes of dipeptidyl peptidase IV (CD26) inhibition
by oligopeptides derived from the N-terminus of HIV-1 Tat indicate
at least two inhibitor binding sites
Susan Lorey
1
, Angela Sto¨ ckel-Maschek
1
,Ju¨ rgen Faust
1
, Wolfgang Brandt
2
, Beate Stiebitz
1
,
Mark D. Gorrell
3
, Thilo Ka¨ hne
4
, Carmen Mrestani-Klaus
1
, Sabine Wrenger
5
, Dirk Reinhold
5
,
Siegfried Ansorge
6
and Klaus Neubert
1
1
Department of Biochemistry/Biotechnology, Institute of Biochemistry, Martin-Luther-University Halle-Wittenberg, Halle, Germany;

2
Institute of Plant Biochemistry, Leibniz Institute Halle, Germany;
3
AW Morrow Gastroenterology and Liver Center,
Royal Prince Alfred Hospital, Newtown NSW, Australia;
4
Department of Internal Medicine, Institute of Experimental
Internal Medicine and
5
Institute of Immunology, Otto-von-Guericke-University Magdeburg, Germany;
6
IMTM Magdeburg, Germany
Dipeptidyl peptidase IV (DP IV, CD26) plays an essential
role in the activation and proliferation of lymphocytes,
which is shown by the immunosuppressive effects of syn-
thetic DP IV inhibitors. Similarly, both human immuno-
deficiency virus-1 (HIV-1) Tat protein and the N-terminal
peptide Tat(1–9) inhibit DP IV activity and T cell prolifer-
ation. Therefore, the N-terminal amino acid sequence of
HIV-1 Tat is important for the inhibition of DP IV.
Recently, we characterized the thromboxane A2 receptor
peptide TXA2-R(1–9), bearing the N-terminal MWP seq-
uence motif, as a potent DP IV inhibitor possibly playing a
functional role during antigen presentation by inhibiting T
cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-
Klaus, C., Fengler, A., Sto
¨
ckel-Maschek, A., Lorey, S.,
Ka
¨

hne, T., Brandt, W., Neubert, K., Ansorge, S. & Rein-
hold, D. (2000) J. Biol. Chem. 275, 22180–22186]. Here, we
demonstrate that amino acid substitutions at different
positions of Tat(1–9) can result in a change of the inhibition
type. Certain Tat(1–9)-related peptides are found to be
competitive, and others linear mixed-type or parabolic
mixed-type inhibitors indicating different inhibitor binding
sitesonDPIV,attheactivesiteandoutoftheactivesite.
The parabolic mixed-type mechanism, attributed to both
non-mutually exclusive inhibitor binding sites of the enzyme,
is described in detail. From the kinetic investigations and
molecular modeling experiments, possible interactions of the
oligopeptides with specified amino acids of DP IV are sug-
gested. These findings give new insights for the development
of more potent and specific peptide-based DP IV inhibitors.
Such inhibitors could be useful for the treatment of auto-
immune and inflammatory diseases.
Keywords: DP IV; CD26; HIV-1 Tat; parabolic inhibition;
mixed-type inhibition.
Dipeptidyl peptidase IV (DP IV, CD26, EC 3.4.14.5) is a
membrane-bound serine protease first identified in rat
kidney [1]. The enzyme occurs in most mammalian epithelial
tissues, such as kidney, liver and intestine [2,3]. DP IV
catalyzes the cleavage of dipeptides from the N-terminus of
oligopeptides and polypeptides provided the penultimate
residue is proline [4]. In the immune system DP IV is an
activation marker of T lymphocytes and is also expressed on
B lymphocytes and NK cells [5–7]. A contribution to signal
transduction processes is ascribed to DP IV by various
authors [8–11]. Furthermore, the enzyme functions as a

binding molecule for adenosine deaminase [12]. The
DP IV-catalyzed hydrolysis of the N-terminus of different
chemokines resulting in changed receptor binding potentials
reflects the importance of the enzymatic activity of DP IV in
humans [13,14]. DP IV inhibitors are currently tested by
different laboratories and companies as therapeutics in
diseases such as diabetes and multiple sclerosis [15,16].
The human immunodeficiency virus-1 transactivator Tat
(HIV-1 Tat, 86 amino acids) is a protein encoded by the
HIV-1 genome. Tat is an intracellular protein playing an
essential role in transactivation of viral genes and in viral
replication [17]. HIV-infected T cells release Tat into the
culture supernatant [18]. Addition of Tat to the cell culture
medium induces a number of immunosuppressive effects,
such as the inhibition of antigen-, anti-CD3- and mitogen-
induced lymphocyte proliferation [19,20]. The mediation of
these inhibition effects may be achieved via the interaction
of Tat with cell surface proteins, for instance DP IV. In
concordance with this finding, Gutheil et al.[21]showed
that Tat binds with high affinity to DP IV and functions as
a potent inhibitor of the enzyme, indicating the possible role
Correspondence to K. Neubert, Department of Biochemistry/
Biotechnology, Institute of Biochemistry, Martin-Luther-University
Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle, Germany.
Fax: + 49 345 5527011, Tel.: + 49 345 5524800/5524849,
E-mail:
Abbreviations: G-CSF, granulocyte colony stimulating factor; HIV-1,
human immunodeficiency virus-1; IL, interleukin; pNA, p-nitroani-
lide; R110, rhodamine 110; DP IV, dipeptidyl peptidase IV;
TXA2-R, thromboxane A2 receptor.

Enzyme: Dipeptidyl peptidase IV (DP IV, CD26, EC 3.4.14.5).
(Received 9 September 2002, revised 10 February 2003,
accepted 13 March 2003)
Eur. J. Biochem. 270, 2147–2156 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03568.x
of Tat–DP IV interactions in AIDS. The immunosuppres-
sive effects of specific DP IV inhibitors and Tat are similar
[20]. We found that the MXP motif of the N-terminal region
of Tat is an important sequence for inhibitory activity,
showing the inhibition of DP IV-catalyzed hydrolysis of
IL-2(1–12) and the inhibition of mitogen-induced prolifer-
ation of human T cells by Tat(1–9) [22]. Amino acid
substitutions at positions 5 and 6 of Tat(1–9) resulted in a
weakening of the immunosuppressive effects reflecting the
importance of the amino acid sequence out of the N-terminal
MXP motif [23]. Recently, we characterized the N-terminus
of thethromboxane A2 receptor, TXA2-R(1–9), as a possible
endogenous inhibitory ligand of DP IV with N-terminal
MWP sequence [24].
To gain further insight into the molecular mechanisms of
Tat–DP IV interactions and thereby contributing to the
understanding of the functional effects mediated by signal
transduction processes induced by Tat, kinetic investiga-
tions mainly of Tat(1–9)-derived peptides as inhibitors of
DP IV were carried out. It is shown that the type of
inhibition is determined not only by the N-terminal amino
acid motif XXP as we discussed earlier [25] but also by the
subsequent amino acids. The inhibition of DP IV by the
peptides Tat(1–9), Trp1-Tat(1–9), Gly3-Tat(1–9) and Ile3-
Tat(1–9) that follows a rarely described parabolic mixed-
type mechanism is presented in detail. Furthermore, the

improvement of the inhibitory potency of oligopeptides
containing a tryptophan residue in position 2 is demonstra-
ted, and we made an effort to explain this by docking studies
based on a model of the C-terminal domain of DP IV. We
present here the first evidence for the existence of at least
two different inhibitor binding sites on DP IV, one in the
catalytic site and the other outside the catalytic site. This
could be important for the development of new, more
effective DP IV inhibitors.
Experimental procedures
Synthesis of oligopeptides
All nonapeptides and Met-IL-2(1–12) (Table 1) were syn-
thesized by solid-phase synthesis with Fmoc technique using
a peptide synthesizer 433A (Applied Biosystems). The
tripeptides MWP and MWV were prepared by solution
synthesis. All peptides were purified by reversed-phase
HPLC and analyzed by mass spectrometry,
1
HNMR
spectroscopy and elemental analysis. The chromogenic
DP IV substrates Ala-Pro-pNA [4] and Gly-Pro-R110-
CO-(CH
2
)
4
Cl [26,27] were synthesized according to standard
procedures of peptide synthesis and were purified by HPLC.
The synthesis and characterization of the DP IV inhibitor
Pro-Pro
(P)

[OPh-4 CL]
2
has been described earlier [28].
Enzyme purification
Human soluble DP IV was produced recombinantly in
CHO cells [13]. The cell culture supernatant of the
transfected cells was applied on a FPLC POROS HQ ion
exchange column and eluted with an increasing gradient of
NaCl. DP IV-containing fractions were subsequently ana-
lyzed by PAGE (silver stained) and the fractions without
any contaminations were pooled for further use.
Enzymatic assay
All enzymatic assays were performed in 0.04
M
Tris/HCl
buffer (pH 7.6, I ¼ 0.125
M
withKCl)at30 °C. The number
of the active sites of DP IV was determined by incubating
DP IV with different concentrations (10
)9
M
to 10
)8
M
)of
the irreversible DP IV inhibitor Pro-Pro
(P)
[OPh-4 CL]
2

for 12 h at 30 °C. After completion of inactivation the
Table 1. Kinetic constants of the inhibition of DP IV-catalysed hydrolysis of Ala-Pro-pNA and Gly-Pro-R110-CO-(CH
2
)
4
Cl. Kinetic constants were
determined by coincubation of at least six different inhibitor concentrations and six different substrate concentrations. The enzymatic assays
contained 0.04
M
Tris/HCl buffer (pH 7.6, I ¼ 0.125), 4.04 · 10
)8
M
DP IV and were incubated at 30 °C. The hydrolysis of Ala-Pro-pNA was
measured by monitoring the released p-nitroaniline at 390 nm. The hydrolysis of Gly-Pro-R110-CO-(CH
2
)
4
Cl was measured by monitoring the
released R110-CO-(CH
2
)
4
Cl at 494 nm. The kinetic constants were evaluated using slope and y-axis-intercept replots of the Dixon plot and/or
Lineweaver–Burk plot.
Compound Amino acid sequence K
i
(
M
) acd Type of inhibition
Tat(1–9)

MDPVDPNIE 2.67 · 10
)4
8.9 0.3 6.5 Parabolic mixed-type
Tat(1–9)
a
MDPVDPNIE 2.30 · 10
)4
0.8 0.8 2.2 Parabolic mixed-type
Trp1-Tat(1–9)
WDPVDPNIE 1.50 · 10
)4
46 1.5 15 Parabolic mixed-type
Gly3-Tat(1–9)
MDGVDPNIE 4.87 · 10
)4
3.7 0.3 2.2 Parabolic mixed-type
Ile3-Tat(1–9)
MDIVDPNIE 1.75 · 10
)3
1.7 9.2 0.01 Parabolic mixed-type
Lys2-Tat(1–9)
MKPVDPNIE 4.27 · 10
)5
10 Linear mixed-type
Trp2-Tat(1–9)
MWPVDPNIE 2.12 · 10
)6
16 Linear mixed-type
Trp2-Tat(1–9)*
MWPVDPNIE 1.70 · 10

)6
4.8 Linear mixed-type
Met-Trp1-G-CSF(1–8)
MWPLGPASS 1.24 · 10
)5
16 Linear mixed-type
Met-IL-2(1–12)
MAPTSSSTKKTQL 2.69 · 10
)4
9.4 Linear mixed-type
Met-Trp-Val
MWV 2.00 · 10
)4
15 Linear mixed-type
Trp2,Ile3-Tat(1–9)
MWIVDPNIE 4.36 · 10
)5
Competitive
TXA2-R(1–9)
MWPNGSSLG 5.02 · 10
)6
Competitive
Met-Trp1-IL-2(1–8)
MWPTSSSTK 1.59 · 10
)5
Competitive
Met-Trp-Pro MWP 2.45 · 10
)5
Competitive
a

Gly-Pro-R110-CO-(CH
2
)
4
Cl was used as substrate.
2148 S. Lorey et al. (Eur. J. Biochem. 270) Ó FEBS 2003
hydrolysis of 10
)4
M
Gly-Pro-pNA was measured by moni-
toring the released p-nitroaniline (pNA) at 390 nm over
120 s. The DP IV concentration in the assay was obtained
from the graph of initial velocity vs. the inhibitor concentra-
tion as the intersection of the regression line with the x-axis.
The DP IV activity was determined using Ala-Pro-pNA
or Gly-Pro-R110-CO-(CH
2
)
4
Cl as substrates. The inhibi-
tion of the hydrolysis of the substrates in at least five
different concentrations (10
)5
M
to 8 · 10
)5
M
)inthe
absence and presence of different inhibitor concentrations
around the expected K

i
values was analyzed by detecting the
enzymatically released pNA (e
390 nm
¼ 11 500
M
)1
Æcm
)1
[4]) or R110-CO(CH
2
)
4
Cl (e
494nm
¼ 29 653
M
)1
Æcm
)1
[27]),
respectively. The measurements were carried out on a
Beckmann DU-650 UV/VIS spectrophotometer. The reac-
tion was started by adding the enzyme (4.04 · 10
)8
M
)and
was run in duplicates over 90 s.
Evaluation of kinetic constants
The kinetic data were calculated using the software

MICRO-
CAL ORIGIN
4.10 and
SIGMAPLOT
5.0.
First, steady state kinetics were analyzed using Eqn (1) for
the Dixon plot where K
i
is the binding constant of the
inhibitor to the noncompetitive site on the enzyme, whilst a
is the factor relating the difference in affinity of the inhibitor
for the same site in the enzyme-substrate complex [29].
1
v
¼
1 þ
aÁK
m
S½

aÁK
i
ÁV
max
Á I½þ
1
V
max
1 þ
K

m
½S

ð1Þ
In the case of linear behavior the distinction between
competitive and linear mixed-type inhibition as well as the
determination of the K
i
valueandthefactora was carried
out using the replot of slopes vs. 1/[S] (Eqn 2). For
competitive inhibition a straight line goes through the origin
whereas for linear mixed-type inhibition the y-axis intercept
is greater than zero [29].
slope ¼
K
m
K
i
ÁV
max
Á
1
½S
þ
1
aÁK
i
ÁV
max
ð2Þ

In the case of parabolic behavior of the Dixon plot the data
were plotted according to Lineweaver–Burk (Eqn 3),
yielding straight lines without a common point of intersec-
tion. For the calculation of the K
i
valueandthefactorsa, c
and d the slopes and intercepts were replotted vs. [I]
according to Eqns (4) and (5), respectively. Here, cÆK
i
represents the competitive inhibition constant.
1
v
¼
K
m
V
max
1 þ
½I
c
1þc

ÁK
i
þ
½I
2
cÁdÁK
2
i

0
@
1
A
Á
1
½S
þ
1
V
max
1 þ
½I
aÁK
i

ð3Þ
slope
LineweaverÀBurk
¼
K
m
V
max
1
½I
þ
1
c
1þc


ÁK
i
þ
½I
cÁdÁK
2
i
0
@
1
A
Á½I
ð4Þ
intercept
LineweaverÀBurk
¼
1
V
max
þ
1
V
max
ÁaÁK
i
Á½Ið5Þ
Molecular modeling
A model of the C-terminal region containing the catalyti-
cally active domain of DP IV has been developed by us and

was described previously [30]. Based on this structural
model we intended to investigate possible docking arrange-
ments of Tat(1–9) and Trp2-Tat(1–9) with DP IV in the
presence of the substrate Ala-Pro-pNA located at the active
site. The molecular graphics program
SYBYL
(TRIPOS
Associates Inc.) with a slightly modified TRIPOS force field
[31] was used. The parameters e of the van der Waals force
field term of all carbon atoms were increased by 0.2 kcalÆ
mol
)1
. The nonbonded cut-off was set to 16 A
˚
. This allows
the application of simulated annealing techniques without
applying a huge water box surrounding the whole enzyme–
ligand complex and periodic boundary conditions. Two
independent simulated annealing runs were carried out. The
first run was started with the solution conformations of
trans Tat(1–9) [23] as well as trans Trp2-Tat(1–9) [24] both
determined by NMR investigations. In the second run a
random-coil conformation was used as starting structure.
Performing 30 cycles of simulated annealing for each run by
heating the system to 700 K within 2000 fs and cooling to
100 K in 2000 fs the ligands do not move far away from the
enzyme at the high temperature, only about 10 A
˚
on
average. During the annealing phase a multitude of stable

docking conformations were obtained. The backbone atoms
of the enzyme were kept fixed. Constraints were applied
between one N-terminal hydrogen atom and one oxygen
atom of the side chain carboxylic group of Glu668 and
between the carbonyl carbon atom of Pro of the substrate
Ala-Pro-pNA and the Ser630 oxygen atom of the enzyme to
hold the substrate inside the active site of DP IV. The
resulting 30 low-temperature docking arrangements of each
run of Tat(1–9) and Trp2-Tat(1–9) were saved in a database
and subsequently minimized with the standard TRIPOS
force field using Gasteiger charges [32] and a distance
dependent dielectric function of e ¼ 4r.
Results
Kinetic analysis of the inhibition of DP IV
Previous investigations have shown that peptides with the
N-terminal MXP sequence inhibit DP IV and suppress
DNA synthesis of peripheral blood mononuclear cells
[22–24]. Our aim was to obtain more information about the
interactions of peptidergic inhibitors with DP IV and the
kinetic mechanisms of inhibition. For that purpose we
investigated the inhibitory effects of Tat(1–9)-derived pep-
tides obtained by amino acid substitutions at positions 1, 2
or 3 of Tat(1–9). Furthermore, a number of other
oligopeptides with the XXP motif were investigated. The
oligopeptides were stable under assay conditions and were
not cleaved enzymatically by DP IV as proved by HPLC.
DP IV retained its full enzymatic activity in the presence of
10
)3
M

oligopeptide solutions as analyzed by dilution
experiments. Therefore, putative loss of DP IV activity
caused by precipitation or inactivation can be excluded. The
K
i
values of the inhibition of DP IV by these compounds
were determined in the range between 10
)6
M
and 10
)3
M
Ó FEBS 2003 DP IV inhibition by XXP containing oligopeptides (Eur. J. Biochem. 270) 2149
(Table 1). Unexpectedly, some structurally highly related
peptides were found to inhibit DP IV according to three
different mechanisms.
The inhibition of DP IV by the N-terminal nonapeptide
of HIV-1 Tat, Tat(1–9), was characterized as parabolic
mixed-type inhibition where two non-mutually exclusive
inhibitor binding sites exist at the enzyme (Fig. 1) [29]. The
inhibitor interacts with the enzyme both at the active site
and at an additional binding site. Binding of one inhibitor
molecule out of the active site, here defined by the K
i
value,
decreases the affinity for binding of the substrate. The
resulting IES complex is catalytically inactive. Binding of
one inhibitor molecule at the competitive site, here defined
by the cÆK
i

value, completely excludes binding of the
substrate. The interaction of a second inhibitor molecule
with the IE complex yielding the IEI complex is character-
ized by the dÆK
i
value. So far, this type of a parabolic
inhibition mechanism has been documented only in a few
publications [33,34]. For DP IV, this inhibition type is
described here for the first time.
Using Ala-Pro-pNA as substrate, a K
i
value of
2.67 · 10
)4
M
and an a value of 8.9, reflecting the increased
affinity of the substrate or the inhibitor to the free enzyme
compared to the EI or ES complex, respectively, were
determined (Table 1). The binding affinities of the inhibitor
to both binding sites, yielding the IE or EI complex, were
only slightly different. On the other hand, the binding
affinity of the second inhibitor molecule to the EI complex
was decreased by a factor of 6.5. The interaction of the
inhibitor with two non-mutually exclusive binding sites at
the enzyme was reflected in the parabolic behavior of the
Dixon plot (Fig. 2A), which therefore was not suitable for
the determination of the K
i
value. From the Hill-plot, binding
of the inhibitor to different binding sites was reflected by the

change of the slope in dependence of the inhibitor concen-
tration. At high inhibitor concentrations a hill coefficient of
)1.6 was determined. The Lineweaver–Burk plot generated
straight lines at different fixed inhibitor concentrations
(1 · 10
)4
M
to 8 · 10
)4
M
). All lines intersected in the
second quadrant but without a common point of intersection
(Fig. 2B). The replot of slopes (slope
Lineweaver–Burk
vs. [I])
produced a parabolic curve (Fig. 2C), the replot of y-axis
intercepts (y-axis intercept
Lineweaver–Burk
vs. [I]) provided a
straight line not going through the origin.
By using the larger substrate Gly-Pro-R110-CO-
(CH
2
)
4
Cl a similar K
i
value for the inhibition of DP IV by
Tat(1–9) was determined (2.30 · 10
)4

M
)whereasthea
Fig. 1. Kinetic model of a parabolic mixed-type inhibition.
Fig. 2. Dixon plot, Lineweaver–Burk plot and slope replot of a parabolic
mixed-type inhibition. (A) Influence of eight fixed concentrations of
Tat(1–9) (1 · 10
)4
M
to 8 · 10
)4
M
) on the hydrolysis of different
substrate concentrations Ala-Pro-pNA (s,1· 10
)5
M
;
h,1.5· 10
)5
M
; n,2· 10
)5
M
; ,,3· 10
)5
M
; e,4· 10
)5
M
; d,
8 · 10

)5
M
) represented as a Dixon plot (1/v vs. [I]). (B) Lineweaver–
Burk plot (1/v vs.1/[S],[I]¼ s,8· 10
)4
M
; h,6· 10
)4
M
; n,
5 · 10
)4
M
; ,,4· 10
)4
M
; e,3· 10
)4
M
; d,2· 10
)4
M
; j,
1 · 10
)4
M
; m, no inhibitor). (C) Slope replot of Lineweaver–Burk plot
(slopes vs. [I]). The reaction mixture contained 0.04
M
Tris/HCl buffer

(pH 7.6, I ¼ 0.125), 4.04 · 10
)8
M
DP IV, and was incubated at
30 °C. The hydrolysis of Ala-Pro-pNA was measured by detecting the
released pNA at 390 nm over 120 s.
2150 S. Lorey et al. (Eur. J. Biochem. 270) Ó FEBS 2003
value and the d value were reduced (Table 1). The K
m
value
for the DP IV-catalyzed hydrolysis of Gly-Pro-R110-CO-
(CH
2
)
4
Cl was estimated as 4.02 · 10
)5
M
(S. Lorey,
unpublished results) indicating a fourfold lower apparent
affinity of the substrate to the active site of DP IV in
comparison to the smaller substrate Ala-Pro-pNA (K
m
1.13 · 10
)5
M
, S. Lorey, unpublished results).
The substitutions of Met1 [Trp1-Tat(1–9)] or Pro3 of
Tat(1–9) [Gly3-Tat(1–9), Ile3-Tat(1–9)] did not change the
inhibition type. The K

i
values of the inhibition of DP IV by
these peptides were in the high micromolar up to the
millimolar range, and using the substrate Ala-Pro-pNA the
a values were a >1 (Table 1). As shown for Tat(1–9), the
binding affinity of a second inhibitor molecule of Gly3-
Tat(1–9) or Trp1-Tat(1–9) to the EI complex was decreased.
On the other hand, in the case of Ile3-Tat(1–9) the
formation of the IEI complex was favored in comparison
to the formation of the EI complex. Moreover, for this
peptide binding to the competitive binding site of DP IV is
diminished (c ¼ 9.2) in comparison to the other peptides.
The peptide containing Trp at position 2, Trp2-Tat(1–9),
turned out to be a stronger DP IV inhibitor than the parent
peptide but inhibited DP IV following a different inhibition
type. Trp2-Tat(1–9) as well as the oligopeptides Lys2-Tat
(1–9), Met-IL-2(1–12), Met-Trp1-G-CSF(1–8) and MWV
were characterized as inhibitors according to the model of
linear mixed-type inhibition [29]. In this case, the inhibitor
and the substrate combine independently and reversibly to
the enzyme, not competing for a common site, forming ES,
EI and IES complexes. The inhibitor is not able to bind to
the competitive binding site. The IES complex is catalyti-
cally inactive. The binding affinity of substrate and inhibitor
to the free enzyme and to the EI or ES complex,
respectively, differs by the factor a.TheK
i
values of the
inhibition of DP IV by these oligopeptides were in the
micromolar range and the a values were in a range between

9.4 and 16 (Table 1) indicating a greater affinity of the
substrate and the inhibitor to the free enzyme compared to
the EI and ES complex, respectively. Figure 3 illustrates the
kinetics of DP IV inhibition by Lys2-Tat(1–9). The Dixon
plot was characterized by straight lines at different fixed
substrate concentrations intersecting in the second quadrant
(Fig. 3A). The replot of slopes represented a straight line
not going through the origin reflecting the linear mixed-type
inhibition (Fig. 3B). In the Lineweaver–Burk plot straight
lines at different fixed inhibitor concentrations (1 · 10
)5
M
to 3 · 10
)4
M
) intersected with a common intersection point
in the second quadrant (not shown).
As shown above for Tat(1–9), the use of the larger
substrate Gly-Pro-R110-CO-(CH
2
)
4
Cl did not affect the K
i
value of Trp2-Tat(1–9) but resulted in a decreased a value.
Interestingly, whereas Trp2-Tat(1–9) bound to the non-
competitive binding site, its N-terminal tripeptide MWP
and Trp2,Ile3-Tat(1–9) exclusively bound at the competitive
binding site. These peptides and the N-terminal peptide
TXA2-R(1–9) of the thromboxane A2 receptor and the

oligopeptide Met-Trp1-IL-2(1–8), all bearing Trp in posi-
tion 2 similar to Trp2-Tat(1–9), were characterized as
competitive inhibitors of DP IV with K
i
values between
5.02 · 10
)6
M
and 4.36 · 10
)5
M
(Table 1). This inhibition
type is characterized by the formation of EI and ES
complexes resulting from a direct competition of the
substrate and the inhibitor molecules for binding at the
active site [29]. The kinetics of DP IV inhibition by TXA2-
R(1–9) is depicted in Fig. 4. The Dixon plot (1/v vs. [I])
provided straight lines at different fixed substrate concen-
trations intersecting in the second quadrant (Fig. 4A). The
replot of slopes (slope
Dixon
vs. 1/[S]) represented a straight
line through the origin characterizing the competitive
inhibition mechanism (Fig. 4B). The Lineweaver–Burk plot
(1/v vs. 1/[S]) yielded straight lines at different fixed inhibitor
concentrations (10
)6
M
to 2 · 10
)5

M
)withacommon
point of intersection on the y-axis at 1/V
max
(not shown).
Docking of Tat(1–9) and Trp2-Tat(1–9) to DP IV
Based on the X-ray structure of the related enzyme prolyl
oligopeptidase [35], which together with DP IV belongs to
Fig. 3. Dixon plot and slope replot of a linear mixed-type inhibition.
(A) Influence of nine fixed concentrations of Lys2-Tat(1–9)
(1 · 10
)5
M
to 3 · 10
)4
M
) on the hydrolysis of five fixed substrate
concentrations Ala-Pro-pNA (s,1· 10
)5
M
; h,1.5· 10
)5
M
; n,
2 · 10
)5
M
; ,,4 · 10
)5
M

; e,8 · 10
)5
M
) represented as a Dixon plot
(1/v vs. [I]). (B) Slope replot of Dixon plot (slopes vs. 1/[S]). The
enzymatic assays contained 0.04
M
Tris/HCl buffer (pH 7.6, I ¼ 0.125),
4.04 · 10
)8
M
DP IV, different inhibitor concentrations and were
incubated at 30 °C. The hydrolysis of Ala-Pro-pNA was measured by
detecting the released pNA at 390 nm over 120 s.
Ó FEBS 2003 DP IV inhibition by XXP containing oligopeptides (Eur. J. Biochem. 270) 2151
the prolyl oligopeptidase family, we constructed a 3D model
of the C-terminal region of DP IV containing the active site
[30]. In order to characterize the noncompetitive binding sites
of Tat(1–9) and Trp2-Tat(1–9) on DP IV, docking studies
with the ES complex on the basis of this 3D model were
performed. Figure 5 illustrates the results of these docking
studies showing the most stable interactions of Tat(1–9) with
this model of DP IV including the substrate Ala-Pro-pNA
bound to the active site with Ala at the S1 and Pro at the S2
binding sites. In the presence of the docked substrate at the
active site some strong interactions of Tat(1–9) with DP IV
could be detected. Salt bridges were formed between the
positively charged N-terminus (Met1) of Tat(1–9) and
Asp709 as well as Asp739 of DP IV, between the C-terminal
Glu9 of Tat(1–9) and the side chains of Arg560 and Lys554 of

DP IV. Furthermore, the side chain of Asp5 of Tat(1–9) was
also able to interact with the side chain of Lys554 of DP IV.
Additionally, hydrogen bonds were formed between the
backbone carbonyl group of Val4 of the peptide and the
backbone amide hydrogen of Ala743 of the enzyme, between
the side chain carbonyl group of Asn7 of Tat(1–9) and the
side chain of Lys554 of DP IV and finally, between the
carbonyl group of Ile8 of the peptide interacting with the
Tyr752 hydroxyl group of the enzyme. A hydrophobic
interactionofthesidechainofMet1ofTat(1–9)withthe
phenyl ring of the substrate Ala-Pro-pNA resulted in a
fixation of the aromatic leaving group.
Rather similar interactions were obtained for docking of
Trp2-Tat(1–9) to DP IV in the presence of the substrate
Ala-Pro-pNA. The salt bridges formed between the ligand
and DP IV were identical to those described above for
Tat(1–9). However, the side chain of Trp2 of this peptide
forms additional hydrophobic interactions with Ile742 of
DP IV. Furthermore, Trp2 interacts with the aromatic
moiety of the substrate.
Discussion
DP IV cleaves oligopeptides at their N-termini by removing
two amino acids, and has a preference for the penultimate
amino acid residue to be proline [4]. Peptides containing
amino acids other than proline in this position (Ala, Gly,
Ser, Thr) are also cleaved but with strongly reduced
efficiency [36,37].
DP IV is not able to catalyze the hydrolysis of peptides
with proline as the third amino acid. In these cases the
oligopeptides function as inhibitors of the enzyme [25,38].

From the biomedical point of view the importance of
DP IV as a costimulatory molecule in T cell activation
processes [8–11], as a hydrolytic enzyme of regulatory
peptides [39] and as an adhesion molecule [12] is well
characterized. Furthermore, it was shown that synthetic
DP IV inhibitors induce immunosuppressive effects result-
ing from the reduction of DNA synthesis and cytokine
production (IL-2, IL-10, IL-12 and IFN-c)ofstimulated
peripheral blood mononuclear cells [11]. Therefore, it was
assumed that DP IV participates in signal transduction
processes. The inhibition of DP IV by the HIV-1 Tat
protein, a viral protein responsible for transactivation of
viral genes, has been shown previously [21,22]. We demon-
strated that the N-terminal amino acid sequence of Tat
represents an important motif for DP IV inhibition [22].
Analogous to synthetic DP IV inhibitors, Tat(1–9) suppres-
ses the DNA synthesis of stimulated peripheral blood
mononuclear cells reflecting the possible role of Tat–DP IV
interactions in AIDS [22]. The function of the viral protein
Tat as an immunomodulatory oligopeptide implies the
existence of soluble or cell surface-expressed endogenous
DP IV-inhibitory molecules. One of them could be the
thromboxane A2 receptor carrying the strong inhibitory
sequence MWP at the N-terminus [24].
The compounds examined in this work are mainly Tat
(1–9)-derived peptides as well as other oligopeptides with
the N-terminal XXP motif. All peptides were characterized
as inhibitors of DP IV. While in earlier studies the
N-terminal XXP was described to be the essential sequence
motif of DP IV inhibitory peptides [25], we found that

oligopeptides with special proline substitutions in the third
Fig. 4. Dixon plot and slope replot of a competitive inhibition. (A)
Influence of eight fixed concentrations of TXA2-R(1–9) (1 · 10
)6
M
to
2 · 10
)5
M
) on the hydrolysis of five fixed substrate concentrations
Ala-Pro-pNA (s,1· 10
)5
M
; h,1.5· 10
)5
M
; n,2· 10
)5
M
; ,,
4 · 10
)5
M
; e,8· 10
)5
M
) represented as a Dixon plot (1/v vs. [I]).
(B) Slope replot of Dixon plot (slopes vs. 1/[S]). The enzymatic assays
contained 0.04
M

Tris/HCl buffer (pH 7.6, I ¼ 0.125), 4.04 · 10
)8
M
DP IV and different inhibitor concentrations and were incubated at
30 °C. The hydrolysis of Ala-Pro-pNA was measured by detecting the
released pNA at 390 nm over 120 s.
2152 S. Lorey et al. (Eur. J. Biochem. 270) Ó FEBS 2003
position are also inhibitors of DP IV, though with lower
potency than known product analogues as DP IV inhibitors
[28,40,41]. Surprisingly, although the tested compounds are
structurally highly related, they differed not only in their K
i
values but also in their inhibition type. Therefore, the
present investigations were focused on the mechanistic
analysis of the inhibition mode of DP IV in order to
obtain a deeper insight into the possible enzyme–inhibitor
interactions based on kinetic measurements and molecular
modeling studies.
For DP IV/CD26 several inhibition modes are known:
competitive, noncompetitive, mixed-type, irreversible, etc.
[28]. In all these cases, the enzyme inhibition takes place by
binding to one site located in or out of the active site. Until
now the inhibition of DP IV/CD26 via binding to two dif-
ferent sites at the enzyme was unknown. This work showed
for the first time that certain peptides may function as DP IV
inhibitors according to a parabolic mixed-type mechanism
that is characterized by the formation of an IEI complex
consisting of one enzyme molecule and two inhibitor
molecules one of them bound in the active site and one of
them at an alternative site. This special, rare type of inhibi-

tion is therefore worth examining although the kinetic con-
stants characterize these inhibitors rather as weak inhibitors.
The Tat(1–9)-related peptides inhibiting DP IV accord-
ing to this in the literature as yet rarely described parabolic
mixed-type mode were characterized by identical amino
acids in position 2 and from positions 4–9 as well as by poor
K
i
values in the range 10
)3
to 10
)4
M
. In comparison to
Tat(1–9), these compounds differ in only one amino acid
position, either position 1 [Trp1-Tat(1–9)] or position 3
[Gly3-Tat(1–9) and Ile3-Tat(1–9)]. The negatively charged
aspartic acid in position 2 seems to disturb binding of the
corresponding peptide to the noncompetitive binding site of
DP IV. In positions 1 and 3, a greater variability of the
amino acids is allowed. Supporting this theory, the Tat(1–
9)-related peptides Lys2-Tat(1–9) and Trp2-Tat(1–9)
derived by substitution of Asp2 inhibited DP IV with
clearly lower K
i
values (10
-5
)10
-6
M

) and according to the
linear mixed-type inhibition mode characterized by inhibitor
binding only to the noncompetitive binding site. There-
fore, the substitution of only one amino acid (Asp2) in the
Tat(1–9) sequence resulted in a change of the inhibition
mode in conjunction with a gain of the ability to bind at the
noncompetitive site.
The determination of the parabolic mixed-type inhibition
mode raised questions according inactivation or precipita-
tion of the enzyme and according enzyme and inhibitor
stabilities under assay conditions. However, HPLC analysis
demonstrated that the inhibitory peptides are not hydro-
lyzed but are stable under test conditions (data not shown).
For DP IV, dilution experiments showed that it retained its
full biological activity at different inhibitor concentrations
thereby excluding precipitation or inactivation. Moreover,
for other structurally related peptidergic inhibitors under
similar assay conditions, more common inhibition modes
were observed suggesting that the measurements for the
inhibitors following parabolic mixed-type mechanism did
not have basic deficiencies, such as enzyme inactivation or
precipitation.
The kinetic data also provide evidence that the range of K
i
values (10
)3
M
to 10
)6
M

) and the different modes of
Fig. 5. Stereo-representation of the interaction of Tat(1–9) with the substrate Ala-Pro-pNA at the active site of a model of DP IV. Carbon atoms are
colored orange [Tat(1–9)], magenta (Ala-Pro-pNA) or gray (DP IV). For clarity only amino acid residues of DP IV essential for the interaction
with the ligands are depicted.
Ó FEBS 2003 DP IV inhibition by XXP containing oligopeptides (Eur. J. Biochem. 270) 2153
inhibition of DP IV by the oligopeptides are not only
affected by the XXP (or XXG, XXI, XXV) sequence motif
but also by the subsequent amino acids. Trp2-Tat(1–9),
TXA2-R(1–9), Met-Trp1-G-CSF(1–8), Met-Trp1-IL-2(1–8)
and the tripeptide MWP contain the identical N-terminal
sequence MWP. Nevertheless, Trp2-Tat(1–9) and Met-
Trp1-G-CSF(1–8) represented linear mixed-type inhibitors,
whereas TXA2-R(1–9), Met-Trp1-IL-2(1–8) and MWP
inhibited DP IV competitively. Therefore, it seems to be
most probable that the MWP motif alone is not responsible
for the inhibition mode especially with regard to the
different subsequent amino acid sequences of these peptides.
On the other hand, the K
i
value seems to be strongly
influenced bythe amino acid in the second position indicating
compounds with tryptophan in this position as the most
potent inhibitors compared to those without tryptophan in
the second position as shown in the present study. Trp2-
Tat(1–9) was the inhibitor with the lowest K
i
value
(2.12 · 10
)6
M

) of all compounds with the N-terminal
XXP sequence tested so far. This inhibition constant is in
the same range as the K
i
values of inhibition of human
recombinant DP IV by the product analogue amino acid
pyrrolidides, e.g. Val-pyrrolidide (K
i
¼ 1.08 · 10
)6
M
)and
Lys[Z(NO
2
)]-pyrrolidide (K
i
¼ 0.42 · 10
)6
M
)(A.Sto
¨
ckel-
Maschek, unpublished results) as well as of the inhibitors
TMC-2A and TSL-225 (K
i
values of 5.3 · 10
)6
M
and
3.6 · 10

)6
M
, respectively), which exert anti-inflammatory
effects on experimentally induced arthritis in rat [42]. The K
i
values of all oligopeptides with the N-terminal MWP motif
were determined to be in the range 10
)6
M
to 10
)5
M
indicating that compounds with tryptophan in position 2
were the most potent inhibitors we examined. Comparing
Tat(1–9) and Trp2-Tat(1–9) by conformational analysis, we
have shown that the backbone conformations of these two
oligopeptides are not significantly altered [24]. Therefore, the
side chain of Trp2 is clearly responsible for the enhanced
inhibitory potency.
Conformational alterations of the peptide backbones
have to be taken into consideration especially in the case of
different amino acid sequences from positions 4–9 in some
peptides. The flexibility of the peptide backbone of Tat(1–9)
is restricted by two proline residues at positions 3 and 6
resulting in a relatively rigid conformation. This is likely to
contribute to the nature of enzyme inhibitor interactions.
Concordantly, all nonapeptides inhibiting DP IV according
to the linear mixed-type mechanism contained both of these
proline residues whereas peptides inhibiting DP IV com-
petitively contained only one proline residue in positions 3

or 6 (Table 1). Comparing inhibition of DP IV by Trp2-
Tat(1–9) with that of Trp2,Ile3-Tat(1–9) it was shown that
the substitution of proline in the third position resulted in a
change of the inhibition mode from a linear mixed-type to a
competitive mechanism.
Using chromogenic substrates such as Ala-Pro-pNA
allowing online measurement of enzymatic hydrolysis, here
we identified Tat(1–9) as a parabolic mixed-type inhibitor
with a K
i
of 2.67 · 10
)4
M
(Table 1). In previous studies, in
a DP IV assay using capillary electrophoresis-based analysis
of the hydrolysis of a more physiological substrate, the
N-terminal peptide IL-2(1–12), Tat(1–9) was found to be a
competitive inhibitor with a K
i
of (1.11 ± 0.12) · 10
)4
M
[23]. One possible explanation for these, on the first view
contradictory results, could be the usage of different
substrates. Therefore, the influence of steric requirements
of different substrates on DP IV inhibition was examined
using the substrate Gly-Pro-R110-CO-(CH
2
)
4

Cl containing
a chain length roughly corresponding to a pentapeptide. In
comparison to the small substrate Ala-Pro-pNA, the larger
substrate Gly-Pro-R110-CO-(CH
2
)
4
Cl did not affect the
type of inhibition and the K
i
value of the oligopeptides
Tat(1–9) and Trp2-Tat(1–9). On the other hand, corres-
ponding to the fourfold difference of the K
m
values of the
hydrolysis of both substrates, the factors a and d were
reduced using Gly-Pro-R110-CO-(CH
2
)
4
Cl. Therefore, in
the presence of the latter substrate, a decreased substrate
affinity resulted in an increased affinity of the inhibitor to
the noncompetitive binding site of the enzyme implying
possible interactions between the ligand and the substrate.
Presumably, because of the definitely shorter chain length of
Gly-Pro-R110-CO-(CH
2
)
4

Cl in comparison to the dodeca-
peptide IL-2(1–12), the different results for DP IV inhibi-
tion by Tat(1–9) obtained with IL-2(1–12) and the
chromogenic substrates could not be explained with Gly-
Pro-R110-CO-(CH
2
)
4
Cl.
In order to examine the binding of inhibitory peptides to
the noncompetitive binding site of substrate-loaded DP IV,
docking studies of Tat(1–9) and Trp2-Tat(1–9) with DP IV
in the presence of the substrate Ala-Pro-pNA, located at the
active site, were carried out on the basis of our 3D model of
the DP IV active site. From this, the preference for interac-
tion of the acidic C-terminus (Glu9) of both peptides with the
basic amino acid residues of DP IV Arg560 and Lys554, as
we postulated earlier [30], was demonstrated. These inter-
actions might be mainly responsible for the binding of
Tat(1–9)-related peptides. Furthermore, it could be demon-
strated that the protonated, positivelycharged N-terminus of
the peptides is able to interact with both Asp709 and Asp739
of DP IV resulting in a considerable stabilization of the
complex. The interactions of the C-terminus as well as the
N-terminus of Tat(1–9)-related peptides permitted the dock-
ing of the inhibitor close to the active site but not directly
inside, thereby allowing the substrate Ala-Pro-pNA to bind
to the active site. However, it can be assumed that the
binding of larger substrates directly interferes with binding of
Tat(1–9). This could be a possible explanation for the

competitive character of DP IV inhibition by Tat(1–9) we
observed in previous studies using the long IL-2(1–12)
substrate [23]. Additionally, multiple interactions of Tat(1–9)
with DP IV contributed to the attractive interaction between
the inhibitor and the enzyme. In the case of Tat(1–9)
containing Asp2, however, this negatively charged residue
was only able to interact with the N-terminus of the peptide
itself but not with the enzyme. Interestingly, a hydrophobic
interactionofthesidechainofMet1ofTat(1–9)withthe
phenyl ring of Ala-Pro-pNA may hinder the DP IV-
catalyzed cleavage of the substrate.
In comparison, the salt bridges between Trp2-Tat(1–9)
and DP IV were identical to those determined for Tat(1–9).
However, the side chain of Trp2 could interact with Ile742.
Furthermore, the indole ring of Trp2 can form strong
hydrophobic interactions with the phenyl ring of Ala-Pro-
pNA resulting in a fixation of the substrate These additional
attractive hydrophobic interactions seem to be responsible
for the improved inhibitory capacity of Trp2-Tat(1–9).
2154 S. Lorey et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Together with former studies, describing competitive
binding of Tat(1–9) directly to the empty active site of
DP IV [23], the docking studies demonstrate the possible
binding of Tat(1–9) to two different binding sites, one
binding site at the active site and one at the noncompetitive
site close to the Ala-Pro-pNA-loaded active site, thus
confirming the results of the inhibition studies with Tat(1–9).
Moreover, the docking studies give a suggestion for the
different inhibition modes observed for Tat(1–9) with
both the chromogenic substrates and the longer substrate

IL-2(1–12).
Trp2-Tat(1–9) exhibiting increased inhibitory capacity
interacts with DP IV at the noncompetitive binding site
close to the active site, and its lower K
i
can be explained by
additional attractive interactions formed between the Trp
side chain and DP IV. Furthermore, the inhibitors stabilize
the leaving group of the bound Ala-Pro–pNA by inter-
actions either with the side chain of Met1 in Tat-(1–9) or
with Trp2 in Trp2-Tat(1–9), thereby hindering the DP IV-
catalyzed cleavage of the substrate.
Very recently, the crystal structure of human DP IV in
complex with the competitive inhibitor valine-pyrrolidide
(Val-Pyr) has been reported [43]. As it was outlined by both
Rasmussen et al. [43] and Gorrell [44], the structure of the
active site of the DP IV model developed by us is in good
agreement with that of the reported crystal structure of
DP IV, particularly concerning the oxyanion hole formed
by Tyr547 together with the backbone NH of Tyr631.
In conclusion, the kinetic investigations presented here
revealed different modes of DP IV inhibition by peptides
with the N-terminal XXP motif. We detected peptides
inhibiting DP IV according to the until now rarely
described parabolic mixed-type mechanism indicating
binding of two inhibitor molecules to two different
binding sites at the enzyme; furthermore, we could show
that single amino acid substitutions at certain positions of
the parent structure alter the mode of inhibition indicating
binding of the peptide to another binding site. In addition

to differences in binding behavior, the compounds varied
in inhibitor potency over three orders of magnitude. The
inhibition of DP IV by the peptides Trp2,Ile3-Tat(1–9),
Gly3-Tat(1–9), Ile3-Tat(1–9) and MWV demonstrated
that the N-terminal XXP sequence is not the essential
structural motif. Tat(1–9)-related peptides with the sub-
stition of Pro3 by other amino acids (Gly, Ile, Val) also
inhibited DP IV, though with lower inhibitory capacity
indicated by higher K
i
values. Furthermore, it was shown
that enzyme–inhibitor interactions depend on multiple
factors such as the amino acid sequence and the
conformation of the peptide backbone of the inhibitor
or specific interactions between the inhibitor and the
bound substrate. On the basis of the active site-containing
3D model of the C-terminal region of DP IV developed
by us [30], evidence for possible molecular interactions of
the inhibitory molecules with DP IV was presented. The
recently reported crystal structure of DP IV [43] provides
a framework for future work and the basis for the
investigation of the protein-bound, pharmacophore con-
formation of the ligands. The stronger inhibitory potency
of MWP-containing peptides [Trp2-Tat(1–9) and TXA2-
R(1–9)] towards DP IV activity and DNA synthesis
among those peptides studied in the present work
underlines the importance of interactions between endog-
enous peptidergic ligands and DP IV, especially with
regard to the role of DP IV in activation and proliferation
of lymphocytes [24]. Our investigations demonstrate for

the first time the existence of different inhibitor binding
sites of DP IV indicating the complex manner of DP IV–
inhibitory peptide interactions and therefore contribute to
the understanding of physiological effects mediated by
Tat(1–9) and its analogs. Additional knowledge of the
molecular mechanisms of inhibitor–DP IV interactions is
important for the development of more potent and more
selective DP IV inhibitors as therapeutics in diseases
including diabetes and multiple sclerosis.
Acknowledgements
Financial support was obtained from the Deutsche Forschungs-
gemeinschaft, SFB 387 and NE 501/2-1, and is gratefully acknowledged.
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