From the National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To better understand how an enzyme controls
cofactor chemistry, we have changed a tryptophan synthase residue that
interacts with the pyridine nitrogen of the pyridoxal phosphate
cofactor from a neutral Ser (-Ser377) to a
negatively charged Asp or Glu. The spectroscopic properties of the
mutant enzymes are altered and become similar to those of tryptophanase
and aspartate aminotransferase, enzymes in which an Asp residue
interacts with the pyridine nitrogen of pyridoxal phosphate. The
absorption spectrum of each mutant enzyme undergoes a
pH-dependent change (pKa ~ 7.7) from a form with a protonated internal aldimine nitrogen
(
max = 416 nm) to a deprotonated form
(
max = 336 nm), whereas the absorption spectra of the
wild type tryptophan synthase
2 subunit and
2
2 complex are pH-independent. The
reaction of the S377D
2
2 complex with
L-serine, L-tryptophan, and other substrates
results in the accumulation of pronounced absorption bands
(
max = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor
chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal
phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid
intermediates.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP)1-dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1, 2). The enzyme protein directs and restricts the catalytic potential of the bound PLP to provide the substrate and reaction specificity and the enhanced reaction rate of the enzyme (3, 4). Thus, it is important to understand how the protein structure controls the cofactor chemistry and enhances catalytic rates.
Information on how the protein structure controls
PLP-dependent reactions is beginning to emerge from x-ray
crystallography and from sequence comparisons designed to establish
evolutionary relationships (5, 6). One of the most important and best studied interactions between the cofactor and the protein active site
of all PLP enzymes is that between the pyridine nitrogen of PLP and an
amino acid side chain (see Fig. 1) (7). In L-aspartate aminotransferase (EC 2.6.1.1) (8) and tryptophanase (EC
4.1.99.1),2 this interaction
is a hydrogen bond/salt bridge between the N-1 proton of PLP and a
negatively charged aspartate side chain. These two enzymes are classed
in the family (5) or fold type I (6). The PLP-binding site of the
tryptophan synthase
subunit (EC
4.2.1.20),3 a representative
of the
family or fold type II, has the neutral hydroxyl of
Ser377 interacting with PLP N-1 (9, 10). Alanine racemase
(EC 5.1.1.1; fold type III) is unique in having a positively charged
arginine near N-1 of PLP (7). Although the enzymes in the three
different fold types have unrelated three-dimensional structures, the
two enzymes in fold type I have related structures. These four enzymes catalyze their different reactions by the pathways illustrated in Fig.
1. The primary reactions of tryptophan synthase and tryptophanase are
the synthesis and degradation of L-tryptophan by
-replacement and
-elimination reactions, respectively.
In this work, we have used site-directed mutagenesis to change
Ser377 to Asp (S377D) or Glu (
S377E) and have
determined the effects of these mutations on some kinetic and
spectroscopic properties. We have reported briefly (11) that mutation
of
-Ser377 to Ala, Asp, or Glu results in a >100-fold
decrease in the rate of conversion of L-serine and indole
to tryptophan and that the
S377D and
S377E
2
2 complexes display some spectral
properties similar to those of tryptophanase and aspartate
aminotransferase.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals and Buffers--
PLP and
-chloro-L-alanine hydrochloride were from Sigma.
L-Serine was purchased from Fluka. Solutions of
-chloro-L-alanine hydrochloride were freshly prepared
and adjusted to pH 7.8 with sodium hydroxide immediately before use.
Buffer A (50 mM sodium Bicine containing 1 mM
EDTA at pH 7.8) was used for spectroscopic studies unless otherwise
specified. The other buffer used was composed of 50 mM
triethanolamine/Bicine (pH 7.8) containing 0.2 M NaCl, 0.2 M CsCl, or 0.2 M KCl.
Mutagenesis-- The expression vector pEBA-10 was used as the template for quick and convenient mutagenesis by megaprimer polymerase chain reaction (PCR) (12). Mutagenic primers used in the construction of the missense mutations were as follows (where base changes are underlined): S377D, 5'-GTC-AAT-CTC-GAT-GGC-CGC-GGA-GT-3'; and S377E; 5'-GTC-AAT-CTC-GAA-GGC-CGC-GGA-GTA-3'. Other primers are described (12). The cloning and expression plasmid for each mutant tryptophan synthase was constructed as described (12). Briefly, each mutagenic primer and PE2 (which contains an XbaI restriction site) were used to amplify the first round of PCR with the pEBA-10 template plasmid using Pfu DNA polymerase (Stratagene). The first PCR fragments were purified and used directly as primers together with the alternate primer PE5 (which contains an SphI restriction site) to amplify the second round of DNA synthesis with the pEBA-10 template plasmid. Deoxyadenosine (dA) was added to the newly amplified second round PCR products by the non-template-dependent activity of Taq polymerase. The second round PCR fragments were purified and directly inserted into the linearized pCRII sequencing plasmid (Invitrogen), which has single 3'-deoxythymidine (dT) residues. After confirmation of the mutated genes by DNA sequencing, the inserted DNA fragment was restricted with SphI and XbaI restriction enzymes (Promega) and ligated into the original parent plasmid (pEBA-10), which had also been digested with SphI and XbaI.
Enzymes--
Escherichia coli CB149 (13), which lacks
the trp operon, was used as a host strain for plasmid
pEBA-10 (12) that expresses the wild type and mutant subunit forms
(S377D and S377E) of the Salmonella typhimurium tryptophan
synthase
2
2 complex. Cultures of the host
harboring wild type or mutant plasmid were grown, and enzyme expression
was induced with isopropyl-1-thio-
-D-galactopyranoside as described (12). Purification of wild type and mutant
2
2 complexes utilized crystallization
from crude extracts followed by recrystallization (14). The amounts of
purified enzymes obtained from 1-liter cultures were 1000 mg (S377D)
and 270 mg (S377E). Analysis of the purified enzymes by
SDS-polyacrylamide gel electrophoresis reveal that the S377D enzyme
contained a lower content of
subunit (~50%) than the wild type
2
2 complex and that the S377E enzyme contained only
subunit (data not shown). The wild type and S377D
2 subunits were obtained by heat precipitation of the
subunit from the
2
2 complex (15).
Plasmid pEBA-4A8 was used to express the wild type
subunit in
E. coli CB149 (12). The
subunit was purified as
described (16) with a slight modification; after DEAE-Sephacel column
chromatography, all fractions were analyzed by SDS-polyacrylamide gel
electrophoresis, and the fractions showing the single band
corresponding to the
subunit were combined and concentrated. A
1-liter culture yielded ~1 g of homogeneous
subunit. Protein
concentrations were determined from the specific absorbance at 278 nm
using Acm1% = 6.0 for the
holo-
2
2 complex,
Acm1% = 6.5 for the
holo-
2 subunit, and
Acm1% = 4.4 for the
subunit (15).
Spectroscopic Methods-- Absorption spectra were measured in a Hewlett-Packard 8452 diode array spectrophotometer thermostatted at 25 °C. Circular dichroism measurements (mean residue ellipticity in degrees cm2/dmol) were made in a Jasco J-500C spectrophotometer equipped with a DP-500N data processor (Japan Spectroscopic Co., Easton, MD).
Data Analysis-- The effects of pH on absorbance at 416 nm were analyzed by Equation 1,
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
![]() |
![]() |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have altered the active site of the tryptophan synthase subunit by changing a residue near the pyridine nitrogen (N-1) of PLP
from a neutral Ser to a negatively charged Asp or Glu as found in
aspartate aminotransferase and tryptophanase
(Fig. 1). The engineered mutant
subunits were expressed in high yield by a
vector that also expresses the wild type
subunit. Purification of
the mutant
subunits by a method that has been used to purify the
wild type
2
2 complex (12) and
2 subunit (14, 16) resulted in a partial loss of
subunit from the S377D
subunit and a complete loss of
subunit
from the S377E
subunit, as described under "Experimental
Procedures." The results suggest that association of the S377D and
S377E
subunits with the
subunit is weaker than that of the wild
type
subunit, as demonstrated below.
|
Effects of pH on the Absorbance and Ellipticity Properties of the
S377D 2 Subunit and
2
2
Complex--
The absorption spectra of the pyridoxal phosphate
cofactor of the tryptophan synthase
2 subunit and
2
2 complex from E. coli (17)
and of the
2
2 complex from S. typhimurium (18) are pH-independent between pH 6 and 10. The
absorption spectra of the S377A
2 subunit and
2
2 complex are also pH-independent (11).
In contrast, the absorption spectra of the S377D
2
subunit (Fig.
2A) and of the S377E
2 subunit (data not shown) exhibit two
pH-dependent absorption bands with maxima at 334 and 416 nm. The sharp isosbestic point indicates that there are only two
significantly populated species involved.
|
Spectroscopic Properties of the S377D
2
2 Complex: Accumulation of Quinonoid
Intermediates--
The wild type
2
2
complex and
2 subunit catalyze
-replacement reactions
with L-serine and indole or
-mercaptoethanol that proceed through a series of PLP intermediates (Fig. 1), which have
characteristic absorption spectra (20). Two quinonoid-type intermediates occur in this pathway, the first
(E-Q1) after removal of the
-proton of
L-serine and the second (E-Q2) after
the
-addition of a nucleophile to the aminoacrylate intermediate
(E-AA). E-Q1 has been observed in
rapid kinetic studies of the
2
2 complex as a transitory intermediate with maximum absorbance at 460 nm (21),
but does not accumulate under equilibrium conditions. E-Q2 accumulates as a stable intermediate under
steady-state conditions in reactions of the wild type
2
2 complex with L-serine and
nucleophiles, including
-mercaptoethanol and indole (17), and with
the product, L-tryptophan (22, 23).
|
|
Subunit Is Required for Tryptophan Quinonoid Formation by the
S377D
2 Subunit--
The S377D
2 subunit
alone forms no quinonoid intermediate from L-tryptophan in
the presence of Cs+ (Fig. 4).
Addition of 0.5-3.5 molar eq of
subunit results in formation of
increasing amounts of the quinonoid intermediate. The inset
shows a plot of the molar absorbance against the
/
subunit molar
ratio. Fit of the data to Equations 3-6 under "Experimental Procedures" gives values of the apparent dissociation constant Kd(
) = 7.0 ± 0.2 µM and of 43,000 ± 160 M
1
cm
1 for the maximum absorptivity at 504 nm. (The higher
value of the maximum absorptivity at 504 nm, 49,200 M
1 cm
1, reported in Table I was
obtained with more freshly prepared enzyme.) Titration of the S377E
2 subunit with the
subunit by the same method gave
values of Kd(
) = 32 ± 3.6 µM and of 1750 ± 60 M
1
cm
1 for the maximum absorptivity. Thus, association of
the S377E
subunit with the
subunit is weaker than that of the
S377D
subunit, consistent with the greater loss of
subunit
during purification of the S377E
subunit from extracts containing
subunit.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments described above were aimed at assessing the
functional role of -Ser377 in the PLP-binding site of
tryptophan synthase and the effects of replacing this residue with a
negatively charged Asp or Glu residue, which is found in several other
PLP-dependent enzymes including aspartate aminotransferase
and tryptophanase (see Fig. 1). Our most important results are that the
mutant enzymes display pH-dependent spectral changes and
exhibit enhanced formation of quinonoid intermediates. We discuss these
results in relation to the chemistry of PLP and
PLP-dependent enzymes.
All known PLP-dependent enzymes bind PLP as an internal
aldimine (Schiff base) with the -amino group of an enzyme lysyl
residue (see E in Fig. 1). Although the equilibria and
absorption spectra of PLP Schiff bases have been investigated in model
systems (26), binding of PLP to the active site of an enzyme affects
the electronic and spectral properties of the PLP derivatives. The
interaction of a residue X with the pyridine nitrogen (N-1)
of PLP influences the electronic state of the cofactor, the equilibrium
distribution between the different reaction intermediates in Fig. 1,
and the pathway of catalysis. Effects of a negatively charged
carboxylate (X = Asp or Glu) near N-1 of PLP are
discussed here with reference to the probable structures of the low and
high pH forms of internal aldimines shown in Fig.
5.
|
A Nearby Carboxylate Raises the pKa of N-1 of PLP-- Interaction of the carboxylate stabilizes the proton on N-1 (Ha) (see Fig. 5) and increases the pKa by 2-5 units (7, 27). Although the position of the proton Ha cannot be determined by crystallography, semiempirical calculations of absorption spectra of aspartate aminotransferase suggest that the N-1-Asp222 pair may be present in both the charged and neutral states, i.e. Ha may be located on the carboxyl or on N-1 (27).
Although the electron-accepting properties of the pyridine ring are enhanced by protonation (7), the presence of a negatively charged Asp near N-1 would reduce the ability of the pyridine ring to attract electrons. Semiempirical calculations of the electronic absorption spectra of PLP derivatives of mitochondrial aspartate aminotransferase (in which X = Asp222) have been carried out using information from x-ray data (28). Calculations of the molecular orbital energy level for PLP in the presence of various protein residues showed that inclusion of Asp222 alone raises the molecular orbital energies by ~3 eV relative to those of PLP. However, inclusion of groups hydrogen-bonded to Asp222 (a water molecule and the His143-Ser139 hydrogen-bonded pair) substantially lowers the molecular orbital energies. Thus, in the case of aspartate aminotransferase, the charge density on active site Asp is modulated by hydrogen-bonding to other groups. The crystal structure of the wild type tryptophan synthaseA Carboxylate near N-1 of PLP Reduces the pKa of the
Schiff Base Nitrogen--
Although the pKa of the
Schiff base nitrogen is usually well over 11 for model Schiff bases
with PLP, a value close to 9.6 is observed for the Schiff base between
valine and N-methyl-PLP, which has a positive charge on N-1
(29). This result provides evidence that the protonated state of the
pyridine nitrogen (N-1) reduces the pKa of the
Schiff base nitrogen by ~2.5 units. The pH-dependent
changes observed with tryptophanase and aspartate aminotransferase have
been attributed to dissociation of the proton (Hb) on the Schiff base
nitrogen (see Fig. 5). Tryptophanase undergoes a
pH-dependent change (pKa = 7.2) from a
protonated form (max = 420 nm) to a deprotonated form
(
max = 337 nm) in the presence of K+ (30).
Aspartate aminotransferase exhibits a pH-dependent
conversion (pKa = 6.7) from a protonated internal
form (
max = 430 nm) to a deprotonated form
(
max = 358 nm) (31, 32). The finding that mutation of
aspartate aminotransferase Asp222 to Ala (D222A) or Asn
(D222N) yields enzymes with pH-independent absorption spectra provides
evidence that Asp222 is indeed responsible for reducing the
pKa of the Schiff base nitrogen (31, 33).
A Carboxylate near N-1 of PLP Promotes Quinonoid
Formation--
According to the generally accepted mechanism of
catalysis by PLP enzymes, formation of the initial enzyme-substrate
intermediate (E-S in Fig. 1) is followed by withdrawal of an
electron pair from the -carbon into the pyridine ring (1). The
electron-accepting properties of the pyridine ring are enhanced by
protonation of the ring nitrogen (N-1), as discussed above. If the ring
nitrogen is protonated, cleavage of the C-
-H bond leads directly to
the quinonoid E-Q1 in Fig. 1 (for structure, see
Fig. 3). Evidence that the presence of a proton or methyl group on N-1
of PLP facilitates quinonoid formation is provided by studies of
quinonoid formation in model and enzymatic systems with pyridoxal and
N-methylpyridoxal (41). Investigations of the spectra of
quinonoids derived from O-methyl-PLP and PLP suggest that
the proton may have migrated from the Schiff base nitrogen to O-3' (41,
42), as shown in the structure in Fig. 3.
Conclusions--
We conclude that replacing tryptophan synthase
subunit Ser377 with a negatively charged Asp or Glu in
the PLP-binding site alters the electron distribution in the PLP
derivatives. Some of the spectroscopic properties of the mutant enzymes
become similar to those of tryptophanase and aspartate
aminotransferase, enzymes that also have an Asp residue near N-1 of
PLP, but have very different overall structures.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Esmond E. Snell, David E. Metzler, and Dagmar Ringe for reading different drafts of this manuscript and for helpful comments and discussions.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry, Uniformed Services
University of Health Sciences, Bldg. B, Rm. 4047, 4301 Jones Bridge Rd., Bethesda, MD 20814.
§ Present address: Dept. of Immunology and Molecular Biology, Toxicology Division, USAMRID, Fort Detrick, Frederick, MD 20702.
¶ Present address: Dept. of Chemistry, Colgate University, 13 Oak Dr., Hamilton, NY 13346-1398.
To whom correspondence and reprint requests should be
addressed: Lab. of Biochemistry and Genetics, NIDDK, Bldg. 8, Rm. 225, 8 Center Dr. MSC 0830, Bethesda, MD 20892-0830. Tel.: 301-496-2763; Fax: 301-402-0240; E-mail: EdithM{at}intra.niddk.nih.gov.
1 The abbreviations used are: PLP, pyridoxal phosphate; Bicine, N,N-bis(2-hydroxyethyl)glycine; PCR, polymerase chain reaction.
2 Preliminary crystallographic data have been reported for tryptophanase (43). The structure of tryptophanase is very similar to that of tyrosine phenol-lyase, which has been reported in more detail (44, 45). Asp223 interacts with N-1 of PLP in the structure of tryptophanase from Proteus vulgaris.
3
The term 2 subunit is used for
the isolated enzyme in solution;
subunit is used for the enzyme in
the
2
2 complex or to describe a specific
residue in the
subunit.
4
The activity of the S377D
2
2 complex is also very low in the
reaction with
-chloro-L-alanine and indole and in
-elimination reactions with L-serine or
-chloro-L-alanine. The activities of the S377D
2
2 complexes are rather insensitive to pH
between 7 and 9.5 (K.-H. Jhee, unpublished results).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|