The effects of a histidine residue on the C-terminal side of an asparaginyl residue on the rate of deamidation using model pentapeptides

Article abstract of DOI:10.1002/(SICI)1520-6017(200006)89:6<818::AID-JPS14>3.0.CO;2-8

The effects of a histidine (His) residue located on the C-terminal side of an asparaginyl (Asn) residue on the rate of deamidation were studied using Gly-Gln-Asn-X-His pentapeptides. The rates of deamidation of the pentapeptides were determined at 37 °C (I = 0.5) as function of pH, buffer species, and buffer concentration. A capillary electrophoresis stability-indicating assay was developed to monitor simultaneously the disappearance of the starting peptides and the appearance of the degradation products. The rates of degradation of the peptides were pH dependent, increasing with pH, and followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-His-His degraded faster than Gly-Gln-Asn-Gly-His, suggesting that the N+1 His residue is catalyzing the deamidation of the Asn residue. The His side chain at these pH values could function as a general acid catalyst, stabilizing the oxyanionic transition state of the cyclic imide intermediate formation. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-His deamidates more rapidly than Gly-Gln-Asn-His-His. The bulk of the side chain of the N+1 His residue versus the N+1 Gly residue apparently inhibits the flexibility of the peptide around the reaction site and, consequently, reduces the rate of the reaction. The significance of this steric hindrance effect of the N+1 His residue on the rate of deamidation was examined further. It was observed that at pH >6.0, Gly-Gln-Asn-His-His undergoes deamidation faster than Gly-Gln-Asn-Val-His. This observation indicated that, at the higher pH values, the N+1 His residue is also acting as a catalyst. Thus, at basic pH, the N+1 His residue influences the rate of deamidation via two opposing effects; that is, general base catalysis and steric interference. The pentapeptide Gly-Gln-Asn-His-His, in addition to undergoing the deamidation reaction, also undergoes bond cleavage at the Asn-His peptide bond. The enhanced rate of Asn-His peptide bond cleavage can be attributed to the general base behavior of the His residue, leading to increased nucleophilicity of the Asn side-chain amide group. Finally, we have shown that the His residue that is two amino acids removed from the Asn, the N+2 position, has little or no effect on the rate of deamidation. (C) 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Full text of DOI:10.1002/(SICI)1520-6017(200006)89:6<818::AID-JPS14>3.0.CO;2-8

The Effects of a Histidine Residue on the C-Terminal Side of
an Asparaginyl Residue on the Rate of Deamidation Using
Model Pentapeptides
CHIMANLALL GOOLCHARRAN,1 LORI L. STAUFFER,1 JEFFREY L. CLELAND,2 RONALD T. BORCHARDT1
1 Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047
2 Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080
Received 18 November 1999; revised 29 February 2000; accepted 2 March 2000
ABSTRACT: The effects of a histidine (His) residue located on the C-terminal side of an
asparaginyl (Asn) residue on the rate of deamidation were studied using Gly-Gln-Asn-
X-His pentapeptides. The rates of deamidation of the pentapeptides were determined at
37 °C (I ס 0.5) as function of pH, buffer species, and buffer concentration. A capillary
electrophoresis stability-indicating assay was developed to monitor simultaneously the
disappearance of the starting peptides and the appearance of the degradation products.
The rates of degradation of the peptides were pH dependent, increasing with pH, and
followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-His-His de-
graded faster than Gly-Gln-Asn-Gly-His, suggesting that the N+1 His residue is cata-
lyzing the deamidation of the Asn residue. The His side chain at these pH values could
function as a general acid catalyst, stabilizing the oxyanionic transition state of the
cyclic imide intermediate formation. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-
His deamidates more rapidly than Gly-Gln-Asn-His-His. The bulk of the side chain of
the N+1 His residue versus the N+1 Gly residue apparently inhibits the flexibility of the
peptide around the reaction site and, consequently, reduces the rate of the reaction. The
significance of this steric hindrance effect of the N+1 His residue on the rate of deami-
dation was examined further. It was observed that at pH >6.0, Gly-Gln-Asn-His-His
undergoes deamidation faster than Gly-Gln-Asn-Val-His. This observation indicated
that, at the higher pH values, the N+1 His residue is also acting as a catalyst. Thus, at
basic pH, the N+1 His residue influences the rate of deamidation via two opposing
effects; that is, general base catalysis and steric interference. The pentapeptide Gly-
Gln-Asn-His-His, in addition to undergoing the deamidation reaction, also undergoes
bond cleavage at the Asn-His peptide bond. The enhanced rate of Asn-His peptide bond
cleavage can be attributed to the general base behavior of the His residue, leading to
increased nucleophilicity of the Asn side-chain amide group. Finally, we have shown
that the His residue that is two amino acids removed from the Asn, the N+2 position,
has little or no effect on the rate of deamidation. © 2000 Wiley-Liss, Inc. and the American
Pharmaceutical Association J Pharm Sci 89: 818–825, 2000
Keywords: deamidation; VEGF (vascular endothelial growth factor); histidine ca-
talysis; protein degradation
INTRODUCTION
Correspondence to: R.T. Borchardt (Telephone: 785-864-
4820; Fax: 785-864-5736; E-mail: rborchardt@ukans.edu
The nonenzymatic deamidation of asparaginyl
(Asn) residues is one of the major chemical deg-
Journal of Pharmaceutical Sciences, Vol. 89, 818–825 (2000)
© 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association
818
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

EFFECTS OF A HISTIDINE RESIDUE ON PENTAPEPTIDE DEAMIDATION
819
radation pathways of peptides and proteins and
has been an area of continuing interest.1–5 Under
slightly acidic to basic pH conditions (pH 5–10),
mechanistic studies suggested that deamidation
of an Asn residue proceeds through a 5-membered
cyclic imide intermediate6–8 (Scheme I). The in-
termediate is formed by intramolecular attack of
the peptide-bond nitrogen of the succeeding
amino acid on the side-chain carbonyl carbon of
Asn in a reaction that releases ammonia.9–12 It is
generally assumed that the reaction pathway for
the formation of the cyclic imide involves ioniza-
tion of the peptide-bond nitrogen, followed by its
nucleophilic attack on the Asn side-chain car-
bonyl carbon.13,14 The transition state of this re-
action is an oxyanionic tetrahedral intermediate;
subsequent proton transfer to the leaving group
from a general acid and the concerted breakdown
of the tetrahedral intermediate generate the
product.14 These cyclic imides are unstable under
slightly acidic to basic pH conditions (pH 5–10)
and spontaneously hydrolyze to form a mixture of
Asp and isoAsp peptides.7–12
Peptide bond cleavage between Asn and the
succeeding residue has also been observed in pep-
tides and proteins.8,15,16 The reaction is believed
to occur through a pathway similar to deamida-
tion, via a cyclic imide intermediate. This cyclic
Scheme 1.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

820
GOOLCHARRAN ET AL.
imide, however, is formed by attack of the side-
chain amide of Asn on the peptide-bond carbonyl
group.
(GQNVH), and L-Gly-L-Gln-L-Asn-L-Gly-L-Gly
(GQNGG) were synthesized by California Peptide
Research, Inc. (Napa, CA). Sodium phosphate
monobasic, sodium phosphate dibasic, and so-
dium chloride were purchased from Sigma
Chemical Company (St. Louis, MO). Deuterium
oxide (99.9 atom % D) was obtained from Cam-
bridge Isotope Laboratories (Woburn, MA). All
other chemicals were of analytical grade and used
as received. The water used in all studies was
from a Millipore MILLI-Q™ water system (Bed-
ford, MA).
It is well documented in the literature that the
amino acid located on the C-terminal side of the
Asn residue has a significant effect on its rate of
deamidation. For example, peptides with amino
acids with bulky side chains on the C-terminal
side of the Asn residue have slower rates of
deamidation.8,11,12 Other physicochemical prop-
erties of the C-terminal residue can also alter the
rate of deamidation. For example, His, Ser, and
Thr residues can enhance the rate of the reaction
by involvement of their respective side chains in
the mechanism.13,17 Using a series of hexapep-
tides, Brennan and Clarke17 argued that at pH
7.4, the presence of a His residue succeeding an
Asn residue (the N+1 position) did not affect the
rate of succinimide formation. However, the N+1
His residue increases the rate of the competing
side-chain amide nitrogen attack on the peptide
backbone, leading to cleavage of the peptide
bond.17
In this study, we are interested in looking at
the effect of a His residue on the C-terminal side
of an Asn over a wider pH range than that studied
by Brennan and Clarke,17 encompassing the His
residue in its protonated and unprotonated
states. The model peptide that we chose for our
study, Gly-Gln-Asn-His-His, constitutes the se-
quence of residues 8–12 of vascular endothelial
growth factor (VEGF). This sequence was chosen
because the predominant degradation pathway
observed for VEGF is the deamidation of the Asn-
10 residue (Cleland JL, Keck R, Jones AJS, un-
published work). This labile Asn residue in VEGF
is followed on the C-terminal side by a His resi-
due, and its rate of deamidation appears to be
greater than that predicted by simply considering
the steric bulk provided by the His side chain. The
kinetics of the deamidation of Gly-Gln-Asn-His-
His and its derivatives, in which the N+1 and N+2
residues are varied, were investigated in aqueous
buffer solutions as a function of pH, buffer spe-
cies, and concentration.
Buffers
Buffer solutions were prepared with sodium phos-
phate monobasic and dibasic for pH values 5.0–
8.0 and glycine for pH values 9.0–10.0. The buffer
concentration range was 0.15–0.05 M. The buffer
solutions were adjusted to a constant ionic
strength of 0.5 M by adding the appropriate
amount of sodium chloride. The buffers were pre-
pared at the experimental temperature, and the
pH value for a given solution remained un-
changed throughout the investigation. An Orion
(420A) pH meter (Beverly, MA) equipped with an
Orion automatic temperature compensation
(ATC) electrode was used to measure the pH
( 0.05) of the buffer solutions.
Kinetic Measurements
All kinetic experiments were carried out in aque-
ous buffer solutions at 37 °C unless otherwise in-
dicated. The pentapeptides were dissolved in the
appropriate buffer solution to yield an initial con-
centration of 1 mM. Aliquots of 200 ␮L were
transferred to 250-␮L vials, which were then
sealed with a screw cap fitted with a butyl rubber
stopper and stored in a 37 °C incubator. At time
zero and predetermined time intervals, a vial was
removed and cooled to room temperature and the
contents were analyzed by capillary electrophore-
sis. All reactions were carried out for three or
more half-lives.
Solvent Isotope Study
MATERIALS AND METHODS
Materials
The kinetic studies in D2O were carried out at pH
5.0 and 8.0 at 37 °C using 0.1 M phosphate buffer
in deuterium oxide. The ionic strength was ad-
justed to 0.5 M by adding a calculated amount of
NaCl. The pD was determined from the pH meter
reading after applying the appropriate correc-
The pentapeptides L-Gly-L-Gln-L-Asn-L-His-L-
His (GQNHH), L-Gly-L-Gln-L-Asp-L-His-L-His
(GQDHH), L-Gly-L-Gln-L-Asn-L-Gly-L-His
(GQNGH), L-Gly-L-Gln-L-Asn-L-Val-L-His
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EFFECTS OF A HISTIDINE RESIDUE ON PENTAPEPTIDE DEAMIDATION
821
tions.18 The kinetic solvent isotope effect (KSIE)
capillary zone electrophoretic method (see Mate-
rials and Methods). A typical electropherogram
showing the separation of GQNHH and its degra-
dation products at pH 7.0 is shown in Figure 1.
The degradation product that elutes immediately
after the starting material (t ס 9.6 min) was iden-
tified as the Asp-containing pentapeptide by com-
paring its retention time with that of an authentic
sample. The second degradation peak at a reten-
tion time of 10.6 min was assigned as the isoAsp-
containing peptide because its molecular weight
as determined by mass spectrometry was identi-
cal to that observed for the Asp degradation prod-
uct (data not shown). Over the pH range 5–10, the
disappearance of the pentapeptides follows
pseudo-first-order kinetics. The first-order rate
constants, kobs, were obtained from semilogarith-
mic plots of peak area versus time.
is expressed as kH O/kD O, where kH O and kD O
2
2
are the rate constants i2n H2O and D2O, respe2c-
tively.
Sample Analysis
An electrophoretic method was developed for the
separation of the pentapeptides and their degra-
dation products. The capillary electrophoresis
(CE) system used was an ISCO Model 3140 Elec-
tropherograph™ (Lincoln, NE). The temperature
of the CE was maintained at 25 °C by a Fisher
Scientific Isotemp Refrigerated Circulator Model
900 (Pittsburgh, PA). A 50 ␮m i.d. fused-silica
capillary (Polymicro Technologies Inc., Phoenix,
AZ) with a total length of 75 cm and an effective
length of 40 cm was used. The separations were
performed with a running buffer of 40 mM so-
dium phosphate monobasic (pH 3.5). A constant
voltage of 25 kV was applied, yielding a current of
30 ␮A. The detection wavelength was 214 nm.
The data were collected and analyzed by an ISCO
ChemResearch Data Analysis System 150 pro-
gram on a 486 SX computer.
In the neutral and basic pH regions, investiga-
tors7–10,19 have previously shown that deamida-
RESULTS AND DISCUSSION
The kinetics of the degradation of the pentapep-
tides GQNXZ (X ס H, G, or V, and Z ס H, or G)
were studied in aqueous buffer solutions at 37 °C
over the pH range 5–10. The disappearance of the
initial peptide and the appearance of degradation
products were monitored by a stability-indicating
Figure 1. Electropherogram for the deamidation of
GQNHH at pH 7.0. Peak 1 is the parent peptide
(GQNHH), and peaks 2 and 3 are degradation products
GQDHH and GQ(isoD)HH, respectively.
Figure 2. Dependence of rate constants of the deami-
dation of GQNHH at 37 °C, I ס 0.5, on the buffer con-
centration and the pH of the solution.
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822
GOOLCHARRAN ET AL.
tion of Asn residues proceeds exclusively through
the formation of a cyclic imide intermediate fol-
lowed by degradation to Asp and isoAsp products.
In this study, incubation of the peptides resulted
in the Asp and isoAsp degradation products in an
approximately 1:3.5 ratio.
Degradation of GQNXH
The kinetics of degradation of the pentapeptides
GQNHH and GQNGH were studied as a function
of pH, buffer species, and concentration. The
rates of deamidation of both peptides increase
with increasing pH, which is consistent with lit-
erature reports.19 In the pH range studied, the
degradation of GQNGH generated the Asp- and
isoAsp-containing peptides as degradation prod-
ucts. However, the degradation of GQNHH also
generated detectable levels of Asn-His peptide
bond cleavage products at pH values >7.0.
The catalytic effects of the buffers were deter-
mined by measuring the rate of deamidation at
constant pH, ionic strength, and temperature
while varying the buffer concentration. The ob-
served pseudo-first-order rate constants, when
plotted against total buffer concentrations, [Bt],
yielded linear plots (Figure 2). The slopes of these
lines represent the second-order catalytic rate
constants, kcat, and the y-axis intercepts are the
buffer-independent rate constants, ko. The results
indicated that both peptides are subject to buffer
catalysis at pH values >7.0.
Figure 4. The pH–rate profile for the deamidation of
GQNGH at 37 °C in aqueous solution. The contribu-
G
tions of the different steps in Scheme III to kobs are
shown.
pH–rate profiles for the deamidation of GQNHH
and GQNGH at 37 °C and an ionic strength of 0.5
M are shown in Figures 3 and 4, respectively.
The pentapeptide GQNHH contains four ioniz-
able groups. However, in the pH range studied,
only three undergo ionization; that is the ␣-amino
group of the N-terminus and the imidizole rings
on the two His residues. The approximate doubly
sigmoidal shape of the profile (Figure 3) suggests
that two of the ionizable groups significantly in-
fluence the kinetics of the reaction. An explana-
tion of the profile is shown in Scheme 2, where
+
AH2 , AH+, and A represent the different ionic
species, all of which may have a different propen-
sities for deamidation.
The shape of the overall pH–rate profile sug-
gests that the rate of deamidation of GQNHH
may be expressed by eq 1:
pH Dependence
The rate constants at zero buffer concentration,
ko, were used to generate the pH–rate profile. The
Hkobs
=
HkofAH
+
HkЈofAH
+
HkЉofA
(1)
++
+
2
where Hko, HkЈo and HkЉo are the microscopic first-
order rate constants for the water catalysis of the
+
+
different ionic species present, and fAH , fAH , and
2
fA represent the fraction of the respective species.
Substituting the fraction of each ionic species of
Figure 3. The pH–rate profile for the deamidation of
GQNHH at 37 °C in aqueous solution. The contribu-
tions of the Hko, HkЈo, and HkЉo steps in Scheme II to
Hkobs are illustrated. The sum of these contributions
gives the solid line.
Scheme 2.
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EFFECTS OF A HISTIDINE RESIDUE ON PENTAPEPTIDE DEAMIDATION
823
Table 1. Calculated Rate Constantsa for the
Deamidation of GQNHH at 37°C
Table 2. Calculated Rate Constantsa According to
Equation 4 for the Deamidation of GQNGH at 37°C
Parameter
Value ( SD)
Parameter
Value ( SD)
Hko
4.53 ( 0.22) × 10−4 h−1
3.86 ( 0.12) × 10−3 h−1
8.13 ( 0.14) × 10−3 h−1
5.49 ( 0.35) × 10−7
GkЈo
3.74 ( 0.28) × 10−4 h−1
6.05 ( 0.13) × 104 M−1 h−1
1.69 ( 0.07) × 103 M−1 h−1
4.36 ( 0.45) × 10−9
HkЈo
HkЉo
Ka1
Ka2
GkЈOH
GkЉOH
Ka
3.55 ( 0.41) × 10−9
a Rate constants are for the ionic species shown in Scheme
III and eq 4.
a Rate constants are for the ionic species shown in Scheme
II and eq 2.
GkЈOH[−OH]fBH is equivalent to GkЉofB. The terms
used in eq 3 were chosen because of the tendency
for deamidation to be a base-catalyzed reaction.
Equation 3 can be rewritten as follows:
GQNHH into eq 1 results in the following equa-
tion:
Hko1bs
=
Hk H+ 2
H+ 2 + Ka H+ + Ka Ka
+
HkЈ Ka H+
+
͔
HkЉoKa1Ka2
ր
͖
͓
o
͔
͓
1
͕
o
Gkobs
=
GkЈ H+ + GkЈOHKw + GkЉOH −OH Ka
ր
͓ ͔ ͓ ͔
͕ ͖
o
(2)
͓
͕
͔
͓
1
͔
͖
2
1
H+ + Ka
(4)
͓
͕
͔
͖
The experimental data were fitted by a least-
squares procedure to eq 2, generating the values
shown in Table 1 for the parameters. The Ka1
value was determined by NMR experiments to
correspond to the ionization constant of the His
imidizole side chain adjacent to the Asn residue.
This assignment was made based on the shift that
occurred in the NMR signal for the C-2 proton on
the imidazole of histidine in the pH range 5–7
(data not shown). The Ka2 value was then as-
signed to the ␣-amino group of the N-terminus.
The pH–rate profile for the deamidation of
GQNGH (Figure 4) suggests the involvement of
an ionizable group in the kinetics of the reaction.
The kinetic model shown in Scheme III could be
proposed to describe this profile. The overall ve-
locity of the degradation of GQNGH at a given pH
can be expressed as:
The best fit of the theoretical pH–rate profile
(solid line in Figure 4) was obtained by using the
values in Table 2 for the parameters of eq 4. The
Ka value was assigned as the ionization constant
of the N-terminal amino group.
Effect of N+1 His Residue
At neutral to basic pH, the amino acid located at
the N+1 position has been shown to have a sig-
nificant effect on the rate of deamidation of an
Asn residue.4,11,12 The results from these studies
suggested that as the steric bulk of the side chain
on the N+1 residue increases, the rate of deami-
dation decreases. To elucidate the effect of the His
G
G
Gkobs = kЈofBH + kЈOH −OH f
+
+
͓
͔
BH
+ GkЉOH −OH f
(3)
+
͓
͔
BH
The term GkЈofBH is kinetically equivalent
to the acid-catalyzed term GkЉHfB. Similarly,
+
Figure 5. The pH–rate profiles for the deamidation of
GQNHH (᭺) and GQNGH (ᮀ) in aqueous solution at
37 °C and I ס 0.5.
Scheme 3.
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824
GOOLCHARRAN ET AL.
Table 3. The Kinetic Solvent Isotope Effect for the
Deamidation of GQNHH and GQNGH at 37°C
more rapidly than GQNHH (Figure 5). The differ-
ences in the rates at higher pH could result from
the greater steric bulk of His side chain, inhibit-
ing flexibility and, consequently, the rate of for-
mation of the cyclic imide. To examine the effects
of steric hindrance of the N+1 residues, we com-
pared the rates of deamidation of GQNHH,
GQNVH, and GQNGH. The His side chain occu-
pies a larger volume than that of Val;22 as such
and based on previous observations, one would
expect the Val analogue to undergo cyclization
faster than the His analogue. However, at all pH
values studied, GQNHH deamidated faster than
GQNVH (Table 4), suggesting that the N+1 His
residue is also acting as a catalyst in the higher
pH region. Thus, at basic pH, the N+1 His residue
influences the rate of deamidation via two oppos-
ing effects, general base catalysis and steric in-
terference.
For the pentapeptide GQNHH, at pH >7.0, de-
tectable levels of Asn-His peptide bond cleavage
products (retention times: t ס 6.8 and 7.2 min)
were observed. This observation is consistent
with that of Brennan and Clarke,17 who also ob-
served spontaneous cleavage of the Asn-His pep-
tide bond at pH 7.4. The enhanced rate of Asn-His
peptide bond cleavage can be attributed to the
ability of the His side chain to hydrogen bond
with the Asn side-chain amide, increasing its nu-
cleophilicity and, thus, the rate of peptide bond
cleavage. The effects of N+2 His residue on the
rate of deamidation were also examined. As
shown in Table 4, the rate of deamidation of
GQNGH is comparable to that of GQNGG. This
noninvolvement of the N+2 His residue in the ki-
netics of the reaction is in agreement with the
pH–rate profile information.
k
/k
H2O D2O
pH
GQNGH
GQNHH
5.0
8.0
1.21
1.12
1.64
1.34
residue at the N+1 position on the rate of deami-
dation of Asn residue, the pH–rate profiles of
GQNHH and GQNGH were compared (Figure 5).
At pH values <6.5, GQNHH degraded slightly
faster than GQNGH, suggesting that the N+1 His
residue is acting as a catalyst in this pH region.
The transition state for the formation of cyclic
imide is assumed to be an oxyanionic tetrahedral
intermediate whose stabilization will result in an
enhancement of the rate of deamidation. Below
pH 6.5, the His side chain can function as a gen-
eral acid group, providing a proton to stabilize the
transition state and, consequently, increasing the
rate of the reaction. In an effort to distinguish
whether the imidazole is facilitating the deproto-
nation of the peptide-bond nitrogen or facilitating
cyclic imide formation by protonation of the leav-
ing group, kinetic studies were conducted in H2O
and D2O at pH 5.0 and 8.0, and the ratios of the
rates kH O/kD O were calculated. The magnitude of
2
the kine2tic solvent isotope effect shown in Table 3
suggests that the proton on the imidizole is hy-
drogen bonded to the transition state, rather than
being involved in a proton transfer step. A similar
general acid catalytic behavior has been proposed
for the side chains of Ser and Thr residues.13,20,21
Above pH 6.5, GQNGH was more sensitive to
changes in the pH of the solution and degraded
In conclusion, these results suggest that Asn-
His sites in peptides and proteins may be highly
Table 4. Summary of the Rate of Deamidation of the GQNXZ
Peptide Derivatives in 100 mM Phosphate Buffer (I ס 0.5) at 37°C
Rate of Deamidation (h−1 × 103)
pH
5.0
6.0
7.0
8.0
9.0
GQNGG
GQNGH
GQNVH
GQNHH
0.385 (0.02)
2.55 (0.21)
18.58 (0.009)
51.76 (3.1)
143.4 (0.51)
268.5 (4.1)
0.358 (0.04)
1.77 (0.14)
12.41 (0.002)
52.97 (2.6)
121.15 (9.2)
293.30 (2.8)
—
—
0.159 (0.01)
1.46 (0.04)
4.56 (0.13)
—
1.96 (0.09)
11.69 (0.17)
22.21 (0.13)
—
10.0
6.72 (0.09)
19.75 (0.04)
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EFFECTS OF A HISTIDINE RESIDUE ON PENTAPEPTIDE DEAMIDATION
825
of peptide degradation. III. Effect of primary se-
quence on the pathways of deamidation of aspa-
raginyl residues in hexapeptides. Pharm Res 7:
787–793.
susceptible to deamidation and peptide bond
cleavage reactions. We found that the His residue
on the C-terminal side of Asn catalyzes the rate of
deamidation in the pH range studied (i.e., pH
5–10).
12. Stephenson RC, Clarke S. 1989. Succinimide for-
mation from aspartyl and asparaginyl peptides as
a model for the spontaneous degradation of pro-
teins. J Biol Chem 264:6164–6170.
ACKNOWLEDGMENTS
13. Bernhard SA, Berger A, Carter JH, Katchalski E,
Sela M, Shalitin Y. 1962. Co-operative effects of
functional groups in peptides. I. Aspartyl-serine
derivatives. J Am Chem Soc 84:2412–2434.
We thank Dr. Richard Schowen for critical read-
ing of and helpful suggestions with the manu-
script and Dr. David Vander Velde for the NMR
analyses. This work was supported by the United
States Public Health Service (GM-088359, GM-
54195).
14. Capasso S, Mazzarella L, Sica F, Zagari A, Salva-
dori S. 1993. Kinetics and mechanism of succin-
imide ring formation in the deamidation process of
asparagine residues. J Chem Soc, Perkin Trans 2
679–682.
15. Capasso S, Mazzarella L, Sorrentino G, Balboni G,
Kirby AJ.1996. Kinetics and mechanism of the
cleavage of the peptide bond next to asparagine.
Peptides 17:1075–1077.
REFERENCES
1. Aswad DW, editors.1995. Deamidation and Isoas-
partate Formation in Peptides and Proteins. Boca
Raton, FL: CRC Press.
2. Kossiakoff AA. 1988.Tertiary structure is a princi-
pal determinant to protein deamidation. Science
240:191–194.
16. Violand BN, Schlittler MR, Toren PC, Siegel
NR.1990. Formation of isoaspartate 99 in bovine
and porcine somatotropins. J Protein Chem 9:
109–117.
17. Brennan TV, Clarke S. 1995. Effect of adjacent his-
tidine and cysteine residues on the spontaneous
degradation of asparaginyl- and aspartyl-contain-
ing peptides. Int J Pept Protein Res 45:547–553.
3. Manning MC, Patel K, Borchardt RT. 1989. Stabil-
ity of protein pharmaceuticals. Pharm Res 6:
903–918.
4. Robinson AB, Rudd CJ. 1974. Deamidation of glu-
taminyl and asparaginyl residues in peptides and
proteins. Curr Topics Cell Regulation 8:247–295.
5. Wright HT. 1991. Nonenzymatic deamidation of
asparaginyl and glutaminyl residues in proteins.
Crit Rev Biochem Mol Biol 26:1–52.
6. Bornstein P, Balian G. 1977. Cleavage at Asn-Gly
bonds with hydroxylamine. Methods Enzymol 47:
132–145.
7. Capasso S, Mazzarella L, Zagari A. 1989. Deami-
dation via cyclic imide of asparaginyl peptides.
Pept Res 2:195–200.
8. Geiger T, Clarke S. 1987. Deamidation, isomeriza-
tion, and racemization at asparaginyl and aspartyl
residues in peptides. J Biol Chem 262:785–794.
9. Clarke S. 1987. Propensity for spontaneous succin-
imide formation from aspartyl and asparaginyl
residues in cellular proteins. Int J Pept Protein Res
30:808–821.
10. Meinwald YC, Stimson ER, Scheraga HA. 1986.
Deamidation of asparaginyl-glycyl sequence. Int J
Pept Protein Res 28:79–84.
18. Schowen KB, Schowen RL. 1982. Solvent isotope
effects on enzyme systems. Methods Enzymol 87:
551–606.
19. Patel K, Borchardt RT. 1990. Chemical pathways
of peptide degradation. II. Kinetics of deamidation
of an asparaginyl residue in a model hexapeptide.
Pharm Res 7:703–711.
20. Tyler-Cross R, Schirch V.1991. Effects of amino
acid sequence, buffers, and ionic strength on the
rate and mechanism of deamidation of asparagine
residues in small peptides. J Biol Chem 266:
22549–22556.
21. Brennan TV, Clarke S.1995. Deamidation and
isoaspartate formation in model synthetic pep-
tides: The effects of sequence and solution environ-
ment. In Aswad, DW, editor. Deamidation and
Isoaspartate Formation in Peptides and Proteins.
Boca Raton, FL: CRC Press, pp 65–90.
22. Ragone R, Facchiano F, Facchiano AM, Colonna G.
1989. Flexibility plot of proteins. Protein Eng 2:
497–504.
11. Patel K, Borchardt RT. 1990. Chemical pathways
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