Origin of the Different pH Activity Profile in Two Homologous Ketosteroid Isomerases*
Young Sung Yun
,
Tae-Hee Lee
¶,
Gyu Hyun Nam
,
Do Soo Jang
,
Sejeong Shin
¶,
Byung-Ha Oh
¶ and
Kwan Yong Choi
||
From the
Division of Molecular and Life Sciences,
the
National Research Laboratory of Protein
Folding and Engineering, and the ¶National
Creative Research Initiative Center for Biomolecular Recognition, Pohang
University of Science and Technology, Pohang 790-784, South Korea
Received for publication, March 3, 2003
, and in revised form, May 6, 2003.
 |
ABSTRACT
|
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Two homologous
5-3-ketosteroid isomerases from
Comamonas testosteroni (TI-WT) and Pseudomonas putida
biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity
below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38,
while PI-WT does not. Based on the structural analysis of PI-WT, the critical
catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring
NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To
investigate the role of Trp-116, we prepared the F116W mutant of TI-WT
(TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic
parameters of those mutants at different pH levels. PI-W116F exhibited
significantly decreased catalytic activity at acidic pH like TI-WT, whereas
TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased
the kcat/Km value by 2.5- to
4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W
clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond
with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results
demonstrate that the activities of both PI-WT and TI-F116W at low pH were
maintained by a tryptophan, which was able not only to lower the
pKa value of the catalytic base but also to
increase the substrate affinity. This is one example of the strategy nature
can adopt to evolve the diversity of the catalytic function in the enzymes.
Our results provide insight into deciphering the molecular evolution of the
enzyme and creating novel enzymes by protein engineering.
 |
INTRODUCTION
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Isozymes carry out the same enzymatic reaction yet exhibit different
properties such as substrate affinity, preference of different coenzyme,
stability, optimal pH, etc. Even if the sequence differences of those isozymes
are evident, the origin of different properties is mostly not clear at the
molecular level. Identification of functional divergence on the mechanistic
basis is important to understand how the enzyme as a molecular machine can be
evolved in the biological system and to rationally modify the enzyme function
with desired physical and catalytic properties
(1). In the post-genomic era,
protein sequences and high resolution protein structures are being accumulated
rapidly, but the remarkable catalytic mechanism of enzymes as well as the
catalytic strategies utilized by nature to evolve new catalytic functions
remain to be understood at the molecular level
(2). The understanding of the
structural basis for different properties of isozymes and their catalytic
diversity could provide the insights into not only discovering the properties
of the enzymes from their protein sequences or structural data but also
creating novel enzymes by protein engineering.
5-3-Ketosteroid isomerase
(KSI,1 EC 5.3.3.1
[EC]
) is
one of the most proficient enzymes catalyzing an allylic isomerization
reaction at a rate comparable to the diffusion-controlled limit by an
intramolecular transfer of a proton (Scheme
1)
(38).
Although KSIs from two different bacterial sources, Comamonas
testosteroni and Pseudomonas putida biotype B, share only 34%
sequence identity, the catalytic residues Tyr-14, Asp-38, and Asp-99 (the
residues are numbered according to C. testosteroni KSI) are well
conserved in the two isomerases
(9,
10). Moreover, the overall
three-dimensional structures are remarkably similar to each other, indicating
that the two KSIs can share the same catalytic mechanism
(1114).
In KSIs, Asp-38 acts as the only catalytic base to abstract a proton from C-4
of the steroid substrate and to transfer the proton to the C-6 position for
the allylic rearrangement of
5-3-ketosteroids
(3,
14).

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SCHEME 1. Catalytic mechanism of KSI. The reaction has been known to proceed
via a dienolate intermediate by a concerted action of three catalytic
residues, Tyr-14, Asp-38, and Asp-99.
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KSI from P. putida (PI-WT) was reported to exhibit different
dependence of Vmax and Km on
pH compared with that from C. testosteroni (TI-WT)
(15). The
Vmax value of PI-WT was not so sensitive to pH as that of
TI-WT, which had been observed before
(15,
16). The pH-rate profiles for
kcat and
kcat/Km were extensively
investigated for TI-WT (17).
The pKa of the catalytically important group on
the free enzyme (pKE) was determined to be 4.57 utilizing
a nonsticky substrate, 5,10-estrene-3,17-dione
(17). The catalytic activity
of TI-WT is supposed to decrease abruptly below pH 5.0 due to the protonation
of the catalytic base, Asp-38. The pKa values of
catalytic residues can be affected by the local microenvironment of the active
site in the enzyme (18). The
environment of Asp-38 in TI-WT is very similar to that in PI-WT as judged by
crystallographic data of both wild-type enzymes
(11,
13,
19). However, there is a
significant difference in that the carboxyl oxygen of Asp-38 is
hydrogen-bonded to NH on the indole ring of Trp-116 only in PI-WT
(Fig. 1B). Trp-116 in
PI-WT is homologously replaced with Phe-116 in TI-WT
(Fig. 1C). The
different electrostatic environments around the catalytic bases in TI-WT and
PI-WT could affect the pKa of Asp-38, resulting
in different pKE values of two homologous KSIs.

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FIG. 1. Three-dimensional structure of KSI. A, ribbon diagram of a
dimeric KSI from P. putida KSI complexed with d-equilenin,
an intermediate analogue. Three catalytic residues and d-equilenin
are shown in ball-and-stick model representation. B,
monomeric structure of the dimeric KSI from P. putida. C, monomeric
structure of the dimeric KSI from C. testosteroni. The molecular
surface of each KSI monomer using the probe of 1.4 Å radius was
generated. Positive and negative charged regions are indicated by
blue and red, respectively. Asp-38 and Trp-116, or Phe-116
are shown in ball-and-stick model representation. The figures were
drawn using the programs WebLab ViewerPro; Accelrys.
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To investigate the role of Trp-116 in PI-WT, we have prepared the F116W
mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F), and
compared their kinetic parameters at various pHs, respectively. The crystal
structure of TI-F116W determined at 2.0-Å resolution exhibited that the
indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen
of Asp-38. Our kinetic and structural results demonstrate that different pH
activity profiles in the two KSIs originate from a single hydrogen bond
between Trp-116 and Asp-38 and that Trp-116 also contributes to the better
binding of the hydrophobic steroid substrate to compensate for the lowered
basicity of the catalytic residue.
 |
EXPERIMENTAL PROCEDURES
|
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Materials5-Androstene-3,17-dione (5-AND), and
5,10-estrene-3,17-dione (5,10-EST) were purchased from Steraloids. Chemicals
for buffer solutions were from Sigma. Oligonucleotides were from Genotech
Inc., Korea. A QuikChange site-directed mutagenesis kit was supplied by
Stratagene. pKK223-3 plasmid was from Amersham Biosciences. Superose 12 gel
filtration column was obtained from Amersham Biosciences.
MutagenesisPhe-116 of TI-WT was replaced with a tryptophan
to make TI-F116W. TI-F116W was prepared using the QuikChange site-directed
mutagenesis kit (Stratagene) and a thermocycler (Minicycler, MJ Research).
pKSI-TI was used as the DNA template
(11). Two primers were
designed to introduce the F116W mutation into the KSI gene of C.
testosteroni. A forward primer was 5'-AGC ATG CGC GCC TTG
TGG GGC GAG AAG AAT ATT-3' and a reverse primer was
5'-AAT ATT CTT CTC GCC CCA CAA GGC GCG CAT GCT-3';
underlined nucleotides represent the ones changed by point mutations.
Recombinant plasmids were introduced into Escherichia coli XL1-Blue
supercompetent cells (Stratagene) and purified by use of QIAprep Spin Miniprep
kit (Qiagen). The entire KSI gene was then sequenced to confirm the intended
mutation only without the change of other sequences in the KSI gene. The W116F
mutation of PI-WT was described previously
(20).
Expression and Purification of KSI ProteinsThe protein was
overproduced in E. coli BL21(DE3) utilizing the pKK223-3 plasmid
containing the wild-type or mutant KSI gene and purified by deoxycholate
affinity chromatography and Superose 12 gel filtration chromatography as
described previously (21). The
purity of the protein was confirmed by a single band on SDS-PAGE. The protein
concentration was determined utilizing the difference extinction coefficient
between tyrosinate and tyrosine at 295 nm as described previously
(22). The accuracy of the
protein concentration was confirmed by the quantitative analysis of the band
on SDS-PAGE by use of an imaging densitometer (Bio-Rad, GS-700) and a software
program provided by its manufacturer (Molecular Analyst/PC).
Steady-state Kinetic AnalysisThe catalytic activities of
the purified KSIs were determined spectrophotometrically using 5-AND as a
substrate according to the procedure described previously
(21). Various amounts of the
substrate were added to a reaction buffer containing 34 mM
potassium phosphate and 2.5 mM EDTA, pH 7.0, at 25 °C. The
concentration of 5-AND was 12, 35, 58, 82, and 116 µM,
respectively. The final concentration of methanol was 3.3 vol%. The initial
reaction rate was obtained within 1 or 2 min after the initiation of the
enzymatic reaction. The fraction of the substrate converted to the product was
below 10% of the substrate applied to the reaction mixture. The reaction was
monitored by measuring the absorbance at 248 nm, using a spectrophotometer
(Shimadzu, UV-2501 PC). Kinetic data were analyzed by use of Lineweaver-Burk
reciprocal plots to obtain kcat and
Km.
pH Activity ProfilesAll the buffers used to determine the
kinetic constants contained 80 mM NaCl to adjust the ionic strength
of the solution. The buffer solution contained 20 mM sodium acetate
for pH 3.84.9, 20 mM sodium MES for pH 5.26.3, or 20
mM potassium phosphate for pH 6.68.0. The procedures for
obtaining kinetic parameters were the same as those for the steady-state
kinetic analysis as described above. The stability of the enzyme was confirmed
by determining the catalytic activity at pH 7.0 after incubating the enzyme in
the respective assay buffer for 2 min. The observed kinetic parameters were
fitted to Equations 1 and
2 to obtain the
pKE and pKES values, respectively, by
nonlinear least-squares analysis utilizing a computer program (Abelbeck
Software, Kaleidagraph version 3.06).
 | (Eq. 1) |
 | (Eq. 2) |
To obtain the pKE and pKES values of
TI-WT, TI-F116W, and PI-WT from the pH activity profile, a nonsticky
substrate, 5,10-EST, was used as described previously
(17). Similarly, the
pKE and pKES values of PI-W116F were
obtained using 5-AND, which can be considered a nonsticky substrate, because
its kcat/Km values are at
least one order of magnitude smaller than that for PI-WT as described
previously (23). To compare
the kinetic parameters of TI-WT and TI-F116W, both 5,10-EST and 5-AND were
used. The plot of
(kcat/Km)obs
versus pH was utilized to determine pKa
for the free enzymes (pKE) and the plot of
(kcat)obs versus pH to determine
pKa for the enzyme-substrate complexes
(pKES)
(18,
27,
28).
Crystallization and Structure DeterminationThe crystal of
TI-F116W was grown at 25 °C from a solution containing 4% polyethylene
glycol 400, 1.6 M ammonium sulfate, and 0.1 M HEPES, pH
7.5, by the hanging-drop method as described previously
(11). The crystal was briefly
immersed in the same solution containing 15% glycerol. Then the crystal was
flash-cooled in the stream of cooled nitrogen gas (100 K) prior to x-ray data
collection. Diffraction data were obtained by using an R-AXIS IV++
image plate with a rotating anode generator (Rigaku, RU300). The crystal
belongs to the space group P3
(1) with unit cell dimensions
of a = 71.525, b = 71.525, c = 103.340 Å, and
= 120°. Data reduction, merging, and scaling were carried out by
use of the programs DENZO and SCALEPACK, as described previously
(25). The structure was
determined by using a molecular replacement program, AMORE, utilizing the
atomic coordinates of TI-WT (PDB code 8CHO
[PDB]
). Further refinement was carried
out by use of the program CNS as described previously
(26).
Circular Dichroism Spectroscopic AnalysisCircular dichroism
(CD) spectra of the enzymes were obtained with a spectropolarimeter (Jasco,
715) equipped with a Peltier-type temperature controller (Jasco, PTC-348WI),
using a quartz cuvette with 0.2-cm path length. The wild-type and mutant
enzymes at 10 µM were preincubated at 25 °C in a buffer
containing 20 mM sodium acetate for pH 3.8, 4.2, and 5.0, 20
mM sodium MES for pH 6.0, or 20 mM potassium phosphate
for pH 7.2. Scans were collected at 0.1-nm intervals with 1-nm bandwidth and
accumulated three to five times. Each spectrum was corrected by subtracting
the spectrum of the solution containing the used buffer and smoothened by use
of a software program provided by the manufacturer of the
spectropolarimeter.
Native Polyacrylamide Gel ElectrophoresisNative PAGE was
carried out as described previously with a slight modification
(23,
24). The wild-type and mutant
enzymes were subjected to electrophoresis in 8% polyacrylamide gels in 50
mM sodium acetate, pH 4.3, or 50 mM HEPES, pH 7.3.
Electrophoresis was carried out at 4 °C to keep the gel from being
overheated by high voltage. During the electrophoresis, the tray buffer was
circulated between two reservoirs to maintain constant pH. Each gel was
pre-run at 150 V for 1 h, after which 1.02.0 µg of the protein in
the respective buffer solution containing 10% glycerol and 0.003% bromphenol
blue was loaded on the gel and run at 100 V.
 |
RESULTS
|
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Kinetic Parameterskcat and
Km values of TI-F116W and PI-W116F were compared
with those of their respective wild-type enzyme
(Table I). The
kcat values of TI-F116W and PI-W116F were lower than those
of TI-WT and PI-WT by 4.42- and 4.46-fold, respectively. The
Km value of PI-W116F was higher than that of
PI-WT by 2.98-fold, whereas the Km value of
TI-F116W was lower than that of TI-WT by 3.14-fold. TI-F116W exhibited the
kcat/Km value nearly
comparable to that of the wild-type, because both
Km and kcat values were
decreased simultaneously at pH 7.0.
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TABLE I Kinetic parameters of WT and mutant KSIs
pKa values for PI-WT, TI-WT, and T1-F116W were
determined with a nonsticky substrate, 5,10-estrene-3,17-dione.
pKa values for PI-W116F were determined with
5-androstene-3,17-dione.
|
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pH Activity ProfilesTo estimate the
pKa values of the catalytic base in two KSIs,
kinetic parameters were determined at various pHs
(Fig. 2). The observed kinetic
parameters, which were fitted to Equations
1 and
2 to obtain the
pKE and pKES values, respectively, are
listed in Table I. The
pKE values of TI-WT were consistent with the previous
results for two substrates, 5-AND and 5,10-EST
(17). The
pKE value in TI-F116W was decreased by 1.05 pH unit
relative to that of TI-WT. At pH 3.8, the
kcat/Km value of TI-F116W was
increased by 2.5-fold and by 4.7-fold for two substrates, 5,10-EST and 5-AND,
respectively, compared with that of TI-WT. The apparent
pKa values of Asp-38 in PI-WT and in PI-W116F
were estimated to be 3.75 and 5.00, respectively, with a significant decrease
of catalytic activity at low pH in PI-W116F
(Fig. 2).

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FIG. 2. pH dependence of kinetic parameters of WT and mutant KSIs. The
lines represent nonlinear least-squares fits of the data to Equations
1 and
2 to obtain the
pKE and pKES values, respectively, as
listed in Table I. 5,10-EST was
used as a substrate for graphs C and D, and for PI-WT in
graphs A and B. 5-AND was used as a substrate for graphs
E and F, and for PI-W116F in graphs A and
B.
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Structural Analysis of TI-F116WTo identify a hydrogen bond
formed by the indole ring NH of Trp-116 in TI-F116W, the crystal structure of
TI-F116W was determined at 2.0-Å resolution. The statistics of the
crystallographic data and refinement are summarized in
Table II. No significant
structural changes were observed in the mutant KSI compared with TI-WT. The
structure of TI-F116W exhibited that the carboxyl oxygen of Asp-38 could form
a hydrogen bond with the indole ring NH of Trp-116, and the distance between
Asp-38 O
1 and Trp-116 N
1 was measured to be 2.87 Å.
Structural comparison of PI-WT, TI-WT, and TI-F116W indicated clearly that the
hydrogen bond formed by the indole ring NH of tryptophan with the carboxyl
oxygen of the catalytic base was the only difference of the environment around
Asp-38 between two wild-type KSIs, and between TI-WT and TI-F116W
(Fig. 3). For simulated
annealing omit map, the structure was disturbed at 1000 K at an initial
refinement stage when the residues, Trp-116 and Asp-38, were omitted. The
electron density map was then calculated with the omission. Trp-116 and Asp-38
exhibited clear electron densities, supporting that the indole ring NH of
Trp-116 can form a hydrogen bond with the carboxyl group of Asp-38
(Fig. 4).

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FIG. 4. Stereoview of the catalytic base of TI-F116W with
2FoFc-simulated
annealing omit electron density map contoured at 1.0 . Residues
Trp-116 and Asp-38, which were omitted from the model, display clear electron
density. A hydrogen bond between Asp-38 and Trp-116 is represented by a
dashed line. The figure was drawn by using the program BobScript and
rendered using Raster3D.
|
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CD Spectra at Different pHsTo investigate the effect of pH
on the conformation of KSIs, far-UV CD spectra were obtained in the pH range
from 3.8 to 7.2 (Fig. 5). CD
spectra were obtained between 200 and 250 nm to estimate the content of the
secondary structure in the protein. TI-WT and TI-F116W exhibited similar
spectra in the pH range from 3.8 to 7.2, respectively, whereas the spectra of
PI-WT and PI-W116F were marginally changed below pH 5.0, indicating that there
were no significant structural changes over the pH range from 3.8 to 7.2 in
all different forms of KSI.

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FIG. 5. Far-UV CD spectra of WT and mutant KSIs at different pH. Spectra of
10 µM KSI were obtained in the buffer solution containing 20
mM sodium acetate for pH 3.8, 4.2, and 5.0, 20 mM sodium
MES for pH 6.0, or 20 mM potassium phosphate for pH 7.2.; pH 3.8
(x), pH 4.2 (solid line), pH 5.0 (dashed line), pH 6.0
(dotted line), and pH 7.2 ( ).
|
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Native PAGEThe ionization state of the catalytic base in
the two KSIs can affect the overall charge of the enzyme, because the
catalytic base, Asp-38, is located in the boundary between the cavity of the
hydrophobic active site and the solvent
(Fig. 1). Mobilities of PI-WT,
PI-W116F, TI-WT, and TI-F116W in the gel electrophoresis were compared at two
different pHs, 4.3 and 7.3 (Fig.
6). PI-WT and PI-W116F had the same mobility at pH 7.3, whereas
PI-W116F migrated more slowly than PI-WT toward the anode at pH 4.3. TI-WT and
TI-F116W also had the same mobility at pH 7.3, whereas TI-WT migrated more
slowly than TI-F116W toward the anode at pH 4.3, indicating that TI-WT and
PI-W116F have less effective negative charges than TI-F116W and PI-WT, because
the carboxylic group of Asp-38 in those KSIs could be protonated at pH
4.3.

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FIG. 6. Native PAGE of WT and mutant KSIs. The direction of migration is
toward the anode at the bottom of the gels at pH 7.3 in A
and B, and at pH 4.3 in C and D. The same samples
were loaded in different lanes as indicated to compare the migration between
the WT and mutant KSIs more precisely.
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 |
DISCUSSION
|
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The analyses of pH activity profiles and crystallographic structures at
high resolution for the wild-type and mutant forms of two homologous KSIs from
two different bacterial sources allowed us to identify the role of Trp-116,
including a hydrogen bond formed by the indole ring NH with the catalytic
base, Asp-38, in PI-WT. A different environment around Asp-38 was found to be
the origin of the different pH activity profiles of the two KSIs. The
carboxylate ion could be destabilized in a relatively hydrophobic region of
the active sites in TI-WT and PI-W116F, resulting in the increase of the
pKa value of Asp-38. In contrast, the
pKa values of Asp-38 in PI-WT and TI-F116W could
be decreased, because the carboxylate ion in Asp-38 was stabilized by a
hydrogen bond with Trp-116 (Fig.
7).

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FIG. 7. Putative ionization states of the catalytic base, Asp-38, in the WT and
mutant KSIs at pH 4.0. The pKa values of
Asp-38 in TI-WT and PI-W116F (A) can be higher than those of Asp-38
in PI-WT and TI-F116W (B).
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Considering the different environment of the critical catalytic base,
Asp-38, in two KSIs, the low pKE value of PI-WT can be
attributed to a hydrogen bond between the carboxylate of Asp-38 and the indole
ring NH of Trp-116. The replacement of Trp-116 with a phenylalanine in PI-WT
to mimic the active-site environment of TI-WT resulted in a significant
decrease of the catalytic activity at low pH, and the W116F mutation increased
the pKE value by 1.25 relative to that of PI-WT. The
increase of pKE in PI-W116F is due to the
pKa increase of Asp-38, because the hydrogen bond
between Trp-116 and the carboxylate of Asp-38 could not be formed. The low
mobility of PI-W116F on native PAGE is the evidence for the protonated state
of Asp-38 at pH 4.3, because the apparent pKa
value of Asp-38 was kinetically estimated to be 5.0 in PI-W116F. The far-UV CD
spectra of PI-WT and PI-W116F between 200 and 250 nm were almost
indistinguishable at low pH, indicating that the enzymes did not undergo any
significant conformational change at low pH. Therefore, the decreased activity
of PI-W116F at low pH originates not from any structural change of the
protein, but from the protonation of the catalytic base.
TI-F116W was prepared to confirm that a hydrogen bond between Trp-116 and
Asp-38 could compensate for the decreased activity of TI-WT at low pH by
stabilizing the ionization state of the catalytic base, Asp-38. As expected,
TI-F116W exhibited no decrease of catalytic activity at low pH and increase of
kcat/Km by 4.7- and 2.5-fold
compared with TI-WT when 5-AND and 5,10-EST were used as substrates at pH 3.8,
respectively. As shown in the crystal structure of TI-F116W, a hydrogen bond
can be formed by the indole ring NH of Trp-116 with a carboxyl oxygen of
Asp-38 (Fig. 3). The hydrogen
bond could cause the pKa value of Asp-38 to be
decreased in TI-F116W. The lower mobility of TI-WT relative to TI-F116W on
native PAGE at pH 4.3 was also consistent with the
pKa values estimated kinetically, reflecting the
change of ionization state of the critical catalytic base, Asp-38. The far-UV
CD spectra of TI-WT and TI-F116W showed almost identical patterns in the pH
range from 3.8 to 7.2, indicating that any significant conformational change
over the pH range did not take place. Hence, the low activity of TI-WT at low
pH is not from the structural change but from the protonation of the catalytic
base, Asp-38.
The importance of the indole ring NH of tryptophan was previously suggested
in its interaction with other residues in the protein
(29,
30). Tryptophan can have
characteristics of both hydrophobicity and hydrophilicity due to the
amphipathic property, which originates from the indole ring in its side chain
(30). The amphipathic
characteristic of tryptophan plays two different roles in the catalysis of
KSIs. One role of tryptophan is that the bulky hydrophobic indole ring
contributes to the better binding of the hydrophobic substrate like steroids.
Compared with TI-WT and PI-W116F, PI-WT and TI-F116W have relatively lower
Km values for the hydrophobic steroid substrate
(Table I). This notion that
Trp-116 contributes to the favorable binding of the steroid is consistent with
the previous result in the characterization of PI-W116F
(20). The other role of
tryptophan is the stabilization of ionized state of the catalytic base
residue, Asp-38, at low pH in the hydrophobic active site of KSI, due to a
hydrogen bond between Trp-116 and Asp-38. Although the hydrogen bond could
decrease the basicity of Asp-38, this adverse effect for catalytic activity
could be overcome by the increased binding affinity to the substrate.
The alteration of pH activity profiles by changing the
pKa value of a catalytic base in an enzyme has
been observed previously
(3133).
The pKa value of the active site histidine was
raised by 0.2 pH unit by acetylation of all surface lysines in trypsin
(31). Shifted pH activity
profiles and higher catalytic activity were obtained by changing the surface
charge of subtilisin using site-directed mutagenesis
(32). The
pKa value of His-64 in the active site of
subtilisin was lowered by 0.4 unit at low ionic strength in the range of
0.0050.01 M by the removal of one surface carboxylate
(33). The lowering of the
pKa by employing the change of the surface charge
is dependent basically on the long range macroscopic electrostatic
interaction, which requires a specific condition of low ionic strength to
unmask electrostatic interaction
(32). During evolution,
enzymes may utilize a long-range electrostatic interaction to regulate the
pKa value of the catalytic residue. However, the
low ionic condition to unmask the electrostatic interaction may not be allowed
in the biological system. In both intracellular and extracellular environments
of a typical mammalian cell, the ion concentration can be higher than 0.1
M (34), and total
ion concentration of the bacterial cell was reported to be comparable to that
of the mammalian cell (35).
The direct regulation by use of the neighboring residue could be more
effective than the indirect regulation through the long range electrostatic
interaction in the aspect that it is relatively independent of ionic strength
of the solution. In this study, we demonstrated that the alteration of the
pKa value of the catalytic residue in a mutant
with higher activity than the wild-type enzyme at low pH could be achieved not
by changing surface charge but by directly changing the microscopic
active-site environment of the catalytic residue utilizing a single hydrogen
bond.
In summary, based on comparative studies using two homologous KSIs on the
role of a tryptophan, we demonstrated that tryptophan could alter the pH
activity profile by both forming the hydrogen bond with the catalytic base and
increasing simultaneously the substrate affinity. PI-WT could employ a kind of
a protective strategy to stabilize the deprotonation state of a catalytic
base, which can play a critical role as a base catalyst at low pH. This
strategy is based on the fine regulation of pKa
of the catalytic base with minimizing a loss of the catalytic activity by
increasing the substrate affinity. The enzyme can adapt to the environment to
maintain catalytic activity at low pH, independent of external biological
buffer during evolution. We assume that this strategy can be found in other
enzymes and applied to the alteration of pH activity profile by protein
engineering.
 |
FOOTNOTES
|
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The atomic coordinates and structure factors (code 1OCV) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
* This research was supported by grants from the programs of the National
Research Laboratory and Creative Research Initiative sponsored by Korea
Ministry of Science and Technology and from Korea Science and Engineering
Foundation and by the Brain Korea 21 project (to Y. S. Y., T.-H. L., and D. S.
J.). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
||
To whom correspondence should be addressed. Tel.: 82-54-279-2295; Fax:
82-54-279-8290; E-mail:
kchoi{at}postech.ac.kr.
1 The abbreviations used are: KSI,
5-3-ketosteroid
isomerase (EC 5.3.3.1
[EC]
); 5-AND, 5-androstene-3,17-dione; 5,10-EST,
5,10-estrene-3,17-dione; CD, circular dichroism; WT, wild-type; MES,
2-(N-morpholino)-ethanesulfonic acid; PI-WT, wild-type KSI from
P. putida biotype B; PI-W116F, W116F mutant of PI-WT; TI-WT,
wild-type KSI from C. testosteroni; TI-F116W, F116W mutant of TI-WT;
KE, ionization constant of the free enzyme;
KES, ionization constant of the enzyme-substrate
complex. 
 |
ACKNOWLEDGMENTS
|
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We thank Yeon-Gil Kim for help in preparing the simulated annealing omit
map.
 |
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