(Received for publication, May 1, 1997, and in revised form, May 21, 1997)
From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
The hydroxyl group of Tyr-78 of yeast guanylate kinase (GK) is hydrogen-bonded to the phosphate of the bound GMP as revealed by x-ray crystallography. The structural and functional roles of Tyr-78 were evaluated by site-directed mutagenesis, kinetics, guanidine hydrochloride-induced denaturation, and nuclear magnetic resonance spectroscopy (NMR). Substitution of Tyr-78 with a phenylalanine resulted in a decrease in kcat by a factor of 131, an increase in Km(GMP) by a factor of 20 and an increase in Ki(GMP) by a factor of 18. Km(MgATP) and Ki(MgATP) were very similar to those of the wild-type (WT) GK. The conformational stability of the mutant was lower than that of the WT by 1.0 kcal/mol as measured by guanidine hydrochloride-induced denaturation. Detailed comparison of the TOCSY and NOESY spectra of the WT GK and the mutant indicated that the conformation of Y78F is little perturbed relative to that of the WT GK at the free state and the conformation of Y78F·GMP complex is also very similar to that of the WT·GMP complex. The results taken together showed that the hydrogen bond between Tyr-78 and GMP stabilizes the GK·GMP complex by 1.7 kcal/mol, the ternary complex by 1.8 kcal/mol, and the transition state by 4.6 kcal/mol. Tyr-78 is not essential for proper folding of the enzyme but it may contribute to the conformational stability. Solvent-accessible aromatic residues were identified by using the paramagnetic probe 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. Comparison of the free and GMP-bound forms of the WT GK by NMR indicated that there are changes in conformation and dynamics upon binding of GMP.
Guanylate kinase (GK)1
catalyzes the reversible phosphoryl transfer from ATP to GMP in the
presence of Mg2+ (1). Like other nucleoside monophosphate
(NMP) kinases, GK plays an important role in the synthesis of the
nucleotide precursors for nucleic acids. Interestingly, several
recently discovered membrane proteins contain domains that share
significant homology with the entire sequence of GK (2). It has been
suggested that GK may be involved in guanine nucleotide-mediated signal
transduction pathways by regulating the ratio of GTP and GDP (3). Human GK plays an important role in activation of the anti-herpes drugs acyclovir and gancyclovir (4, 5) and the anti-HIV agent carbovir (6).
It has been shown that GK has high stereospecificity toward activation
of these drugs (6, 7). Thus it is of biomedical significance to
elucidate the catalytic mechanism of the enzyme and the structural
basis of nucleotide specificity. Human GK has been recently cloned (8,
9). However, the human enzyme is inactive when it is expressed in
Escherichia coli or produced by cell-free translations (9).
We have been interested in studying the structure-function
relationships of yeast GK because its structure has been determined by
x-ray crystallography. Yeast GK shares ~50% amino acid identities
with the human enzyme. The amino acid residues that interact with the
bound GMP (Fig. 1) are strictly conserved
not only between the two enzymes but also among all known GKs.
Recently we have reported the thermodynamic and kinetic properties of the wild-type GK and the roles of Tyr-50 in structure and catalysis (10, 11). In this report we describe characterization of the roles of Tyr-78 by site-directed mutagenesis in conjunction with kinetics, two-dimensional NMR, and chemical-induced denaturation. We show that Tyr-78 plays an important role in binding of GMP and catalysis. It also contributes to the conformational stability of GK but it is not important for appropriate folding. In addition, we have examined the conformational and dynamic changes of the WT GK induced by binding of GMP.
Nucleotides, coupling enzymes, and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were purchased from Sigma. Perdeuterated Tris and L-phenylalanine (ring-D5, 98%) were purchased from CIL. Ultrapure guanidine hydrochloride (GdnHCl) and DNA sequencing kit were purchased from U. S. Biochemical Corp. Affi-Gel Blue gel was purchased from Bio-Rad.
Site-directed Mutagenesis and Protein PurificationThe
oligonucleotide for making the mutant Y78F was
5-GGTAACTACTTTGGTAGTACT-3
. The mutant was generated by the method of Kunkel (12) and selected by DNA sequencing. To ensure that there were
no unintended mutations in the mutant, the entire sequence of the
mutated gene was determined. Expression and purification of the mutant
were the same as described for the wild-type GK (10) except that Y78F
was eluted with 5 mM ATP in the step of Affi-Gel Blue gel
chromatography.
The E. coli
strain DL49PS pLysS (a gift of Dr. David M. LeMaster) was used for
isotopic labeling of GK. The labeling medium was prepared with
L-phenylalanine (ring-D5, 98%) and other
nutrients according to Muchmore et al. (13). The bacterial
cells were grown in the medium containing 100 µg/ml ampicillin and 20 µg/ml chloramphenicol at 37 °C. GK expression was induced by
addition of 0.5 mM
isopropyl-1-thio--D-galactopyranoside (final
concentration) when OD600 reached 0.8. The cells were
harvested after ~3 h of further incubation. The labeled GK was
purified as described previously (10).
The kinetic experiments were carried out by measuring the formation of ADP and GDP using a coupled assay (1, 10). The reaction solution in 1 ml contained 100 mM Tris-HCl, pH 7.7, 100 mM KCl, 5 mM MgCl2, 1.5 mM phosphoenolpyruvate, 0.2 mM NADH, 40 units of pyruvate kinase, 85 units of lactate dehydrogenase, 17 µg of Y78F, and varied amounts of ATP and GMP. The reaction was initiated by addition of Y78F at 25 °C. Kinetic parameters were obtained by nonlinear least square fit of the data to Cleland's equation (14),
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(Eq. 1) |
GMP and MgATP titrations were performed on a Varian VXR 500 MHz NMR spectrometer. The methyl resonance of a methionine residue was followed by one-dimensional NMR as described previously (10). The experimental parameters are given in the legend of Fig. 2. The dissociation constants were obtained by nonlinear least square fit of the data to the equation,
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(Eq. 2) |
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(Eq. 3) |
![]() |
(Eq. 4) |
Two-dimensional Proton NMR Methods
All two-dimensional NMR experiments were performed on a Varian VXR 500 NMR spectrometer operating at a proton frequency of 500 MHz. Protein samples were prepared as described previously (10). The protein concentrations were ~2 mM. For the paramagnetic probe experiments, TEMPOL was added directly to the NMR sample tube from a 2 M TEMPOL stock solution. The ratio of TEMPOL and protein was 10 to 1. DQF-COSY (15), clean TOCSY (16-18), and NOESY (19, 20) spectra were acquired with standard pulse sequences at 27 °C. The mixing times were 45 ms for the TOCSY experiments and 200 ms for the NOESY experiments. All the two-dimensional spectra were acquired in the hypercomplex mode. The spectral width was 6000 Hz in both dimensions. The data were usually acquired with 2048 complex points in the t2 dimension and 256 complex points in the t1 dimension. Spectral processing was carried out on a SGI Indigo 2 workstation using the software NMRPipe (21). The time domain data were apodized with a Gaussian function in t2 and a shifted sine bell function in t1. The t1 dimension was zero filled to 1024 points prior to Fourier transformation. The chemical shifts were referenced to internal sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4.
Equilibrium UnfoldingGdnHCl-induced unfolding was followed by fluorometry and circular dichroism (CD) measurements. Both protein and GdnHCl stock solutions were prepared in 100 mM Tris-HCl buffer, pH 7.7, containing 100 mM KCl. The concentration of the GdnHCl stock solution was determined by measuring its refractive index according to Pace (22). The final protein concentration was 0.125 mg/ml. The protein solutions were allowed to equilibrate at room temperature for ~2 h after mixing with GdnHCl. Fluorescence was measured on a Perkin-Elmer fluorometer with excitation wavelength of 282 nm and emission wavelength of 332 nm. CD spectra were obtained with a Jasco-715 spectropolarimeter using a 1-mm path length cell. Changes in ellipticity at 222 nm were followed to get a transition curve of unfolding. The unfolding data were analyzed by the linear extrapolation method (22). The values for unfolding free energy changes were obtained by nonlinear least square analysis of the entire denaturant titration curves as described by Santoro and Bolen (23).
The steady-state kinetic parameters of Y78F are listed in Table I along with those of the WT GK. The effects of the point mutation on the kinetic properties of GK were rather specific. In comparison with the WT GK, the kcat of Y78F decreased by a factor of 131. The Km(GMP) and Ki(GMP) of the mutant increased by a factor of ~20. The Km(MgATP) and Ki(MgATP) of the mutant were similar to those of the WT GK. The binding properties of Y78F were determined by one-dimensional NMR titration experiments. Representative GMP and MgATP titration curves are shown in Fig. 2. The effects of the mutation on the binding properties of GK were also specific. Thus the affinity of Y78F for MgATP was very similar to that of the WT GK, but the affinity of Y78F for GMP decreased by a factor of 12.
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The structural effects of the
mutation were investigated by NMR. To use NMR to characterize the
structure of a protein, one has to make sequential resonance
assignment. For a protein of the size of GK (20.5 kDa), homonuclear
two-dimensional NMR spectra are rather complicated. Thus our NMR
analysis was focused on the better resolved aromatic spin systems.
Yeast GK contains 19 aromatic residues (one histidine, one tryptophan,
six tyrosines, and 11 phenylalanines). They are more or less evenly
distributed among the ATP- and GMP-binding domains according to the
crystal structure of the GMP complex (24). The aromatic spin systems of
the WT GK were identified by a combination of DQF-COSY (data not shown) and TOCSY. The single tryptophan and all six tyrosine spin systems were
easily identified by deuteration of phenylalaninyl ring hydrogens (data
not shown). Of the 11 phenylalaninyl ring spin systems, 10 could be
identified for the free GK and nine for the GMP complex. The missing
phenylalanyl ring spin systems could be overlapping with other aromatic
spin systems or their signals are too broad to be observed in the
two-dimensional correlated experiments. Surprisingly, the
non-exchangeable ring protons of the histidine residue could not be
identified, presumably because they were broadened somehow and
overlapping with other aromatic protons. The identified aromatic spin
systems are shown in Figs. 3A
and 4A. The chemical shifts of
the aromatic spin systems are listed in Tables
II and III.
The ring spin systems of Tyr-50 and Tyr-78 were sequentially assigned
by comparing the TOCSY spectra of the WT GK with those of Y50F (11) and
Y78F because the NMR spectra of the mutants were very similar to those
of the WT GK. The other aromatic spin systems were tentatively assigned
by comparing the NOE patterns of the GK·GMP complex with the crystal
coordinate of the same complex (24). As shown in Fig.
5A, ~20 interresidue NOEs
can be identified. There are strong intermolecular NOEs between the
ring protons of Tyr-50 and Tyr-78 and H8 of the bound GMP because the distances are only ~3 Å. Yb was tentatively assigned to
Tyr-77 because it has as many as six NOEs with the aromatic protons of
Trp-70 (the six NOE cross-peaks could be clearly identified in the
NOESY spectrum of the GK with deuterated phenylalaninyl rings, data not
shown). According to the crystal structure of the GK·GMP complex,
among the aromatic residues, only the ring protons of Tyr-77 are within
5 Å distance of Trp-70. Yd was assigned to Tyr-25 because only Tyr-25
is close to two phenylalaninyl residues, namely Phe-29 or Phe-183. The
NOE cross-peaks between Fb and Fd could be attributed to Phe-29 and
Phe-183 because only the two phenylalaninyl residues are close to
each other and also to Tyr-25. Fi/Fj was tentatively assigned to Phe-52
or Phe-73 because it has NOE with the aromatic protons of Tyr-50 in the
free GK and the aromatic protons of Tyr-78 in the GMP complex. Solvent
accessibility of the aromatic residues was examined by addition of the
paramagnetic probe 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl
(TEMPOL). The TOCSY spectra of the free and GMP-bound WT GK in the
presence of TEMPOL are shown in Figs. 3B and 4B.
The degree of solvent accessibility was assessed by reductions in the
TOCSY cross-peak volumes caused by TEMPOL. The results are summarized
in the last columns of Tables II and III.
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Comparison of the NMR Properties of the WT GK and Y78F
Substitution of Tyr-78 with a phenylalanine caused few
significant changes in the chemical shifts of the aromatic protons of
the free enzyme. The TOCSY spectra of the free WT GK and Y78F were
almost identical (data not shown). None of the aromatic proton resonances differed by >0.05 ppm between the WT GK and the mutant. The
changes in the aromatic shifts of the GMP-bound form were also limited.
Only seven resonances (five residues) differed by >0.05 ppm between
the WT and the mutant. The largest change in chemical shift (0.30 ppm)
was found in Tyr-50. Conformational changes are best characterized by
NOESY experiments. Comparison of the NOESY spectra of the WT GK and
Y78F is shown in Figs. 5 (free form) and
6 (GMP-bound form). In the free form, all
the interresidue NOEs except one (NOE 9) were observed in both the WT
GK and Y78F. NOE 9 was missing in Y78F. The NOEs between Tyr-78 and
other aromatic residues in the WT GK were replaced by the NOEs between
Phe-78 and other residues in the mutant. In the GMP-bound form, all
interresidue NOEs except four weak ones were observed in both the WT GK
and Y78F. More importantly, the intermolecular NOEs between GK and the
bound GMP were also retained in the mutant. The results indicated that
the conformation of Y78F is very similar to that of the WT GK in both
free and GMP-bound forms.
Conformational Stability of the WT GK and Y78F
GdnHCl-induced
denaturation experiments were used to measure the conformational
stability of the proteins. The unfolding reaction was reversible as
demonstrated by refolding after complete unfolding by GdnHCl. Typical
denaturation curves are shown in Fig. 7.
The results were characteristic of two-state unfolding. Furthermore, the transition curves obtained by fluorometry were very similar to that
obtained by CD measurements (data not shown), suggesting that the
two-state model was appropriate for analysis of the data. The free
energy changes of unfolding obtained by the linear extrapolation method
(22, 23) are listed in Table I. The results indicated that the hydroxyl
group of the phenolic rings of Tyr-78 contributes to the conformational
stability by ~1.0 kcal/mol.
GK belongs to the family of NMP kinases. Among NMP kinases,
adenylate kinase has been most extensively studied (25). High resolution crystal structures have been reported for adenylate kinase,
GK, and UMP/CMP kinase (24, 26-32). The ATP-binding sites of all known
structures are highly conserved. The AMP-binding site of adenylate
kinase and the UMP/CMP-binding site of UMP/CMP kinase are also very
similar. On the other hand, the chain fold of the GMP-binding domain of
GK is grossly different from those of the NMP-binding domains of
adenylate kinase and UMP/CMP kinase (24). The AMP-binding domain and
the UMP/CMP-binding domain are completely -helical. In contrast, the
GMP-binding domain is composed of a four-stranded
-sheet and only a
short helix (Fig. 1). While only two arginine residues are involved in
binding the phosphate moiety of the NMP in adenylate kinase or UMP/CMP kinase, the phosphate moiety of the bound GMP in GK interacts with a
pair of tyrosine residues as well as a pair of arginine residues. In
this study, we have focused on the structural and functional roles of
Tyr-78. Substitution of Tyr-78 with a phenylalanine caused a decrease
in kcat by a factor of 131 and increases in Km and Ki for GMP by a factor of
~20, and little changes in Km and
Ki for MgATP. The mutation also resulted in a
decrease in the stability of the enzyme by ~1.0 kcal/mol. Since the
changes in the kinetic properties and conformational stability could be
due to global structural perturbations, we have compared the
conformation of the mutant with that of the WT GK by NMR. Detailed
analysis of the two-dimensional spectra of the aromatic protons has
indicated that there are only a few small changes (<0.05 ppm) in the
chemical shifts in the mutant spectra at the free state, and all the
interresidue NOEs except a weak one are retained. The results suggest
that Y78F is appropriately folded and its conformation is highly
similar to that of the WT GK. The decrease in conformational stability
is not likely the result of global structural changes. Comparison of
the two-dimensional spectra of the GMP-bound forms has indicated that
there are some large changes in the chemical shifts (Tyr-50 and Fa,
>0.1 ppm). Since both Tyr-78 and Tyr-50 are hydrogen-bonded to the
phosphate of GMP (24), the chemical shift changes of Tyr-50 are likely due to changes in the local electronic environment. Substitution of
Tyr-50 with a phenylalanine also causes large changes in the chemical
shifts of the aromatic protons of Tyr-78 at the GMP-bound state (11).
However, replacement of Tyr-50 or Tyr-78 with a phenylalanine does
not alter the chemical shifts of the other residue in the free form,
indicating the two tyrosine residues do not interact at the unliganded
state. The causes for the chemical shift changes of other residues are
not obvious. Some minor changes in the chemical shifts are likely due
to small differences in the experimental conditions such as pH and
degree of GMP saturation. Most interresidue NOEs observed in the NOESY
spectrum of the WT complex are also observed in the NOESY spectrum of
the mutant complex. Furthermore, the substituted phenylalanine
appears to assume the position of Tyr-78 because Phe-78 in the mutant
complex has NOEs with both Tyr-50 and the bound GMP as Tyr-78 in the WT complex. Taken together, the results suggest that the conformation of
the mutant complex is very similar to that of the WT complex. Therefore
we conclude that the changes in the kinetic parameters are not due to
global conformational changes. This is corroborated by the lack of
significant changes in Km and Ki for MgATP. The changes in the kinetic properties are also unlikely to
be caused by the decrease in conformational stability because the
mutant is still stable for days at room temperature. The above information allows us to attribute the changes in the properties of the
mutant to the removal of the hydrogen bond between the hydroxyl group
of Tyr-78 and the phosphate of GMP. On the basis of the kinetic data we
estimate that the hydrogen bond between Tyr-78 and GMP stabilizes the
binary GMP complex by 1.7 kcal/mol, the ternary complex by 1.8 kcal/mol, and the transition state by 4.6 kcal/mol.
We have reported the characterization of the structural and functional roles of Tyr-50 (11). The hydroxyl group of Tyr-50 is also hydrogen-bonded to the phosphate of GMP with similar geometry. In comparison with Try-50, Tyr-78 contributes less to the stabilization of the binary and ternary complexes by ~0.5 kcal/mol. This is consistent with the crystallographic observation that the hydrogen bond between Tyr-78 and the phosphate of GMP is slightly longer and therefore a bit weaker than that between Tyr-50 and the phosphate of GMP. However, compared with Tyr-50, Tyr-78 contributes more to the transition state stabilization by 1.4 kcal/mol and therefore is more important for catalysis. This suggests that the hydrogen bond between Tyr-78 and GMP becomes stronger than that between Tyr-50 and GMP as the reaction progresses to the transition state.
Substrate-induced fit is believed to be the mechanism by which kinases
use to avoid hydrolytic activity (33). It has been suggested that the
active centers of NMP kinases are assembled with large domain movements
upon binding of both substrates (31). GK is also expected to undergo
substrate-induced movements (24). Because of its small size and
stability, GK is an excellent model system for detailed analysis of the
mechanisms of substrate-induced fit by NMR. Binding of GMP causes
significant changes (>0.05 ppm) in the chemical shifts of a number of
aromatic spin systems (Tyr-50, Trp-70, Tyr-78, Ya, Fa, Fc, and Fi/Fj)
(Figs. 2A and 3A). Fe changes so dramatically
that it cannot be identified in the spectra of the complexed form. The
changes in the chemical shifts of Tyr-50 and Tyr-78 are likely caused
by hydrogen bonding to the phosphate of the bound GMP and the ring
current effects of GMP. The chemical shift changes of other residues
may reflect conformational changes induced by binding of GMP, although
small chemical shift changes may be due to slight variations in the
experimental conditions. These chemical shift changes are unlikely due
to ring current effects because all aromatic residues except Tyr-50 and
Tyr-78 are 9 Å away from the guanine moiety of GMP. The
interpretation is supported by the NOESY data and the paramagnetic
probe TEMPOL. Comparison of the NOESY spectra of the free and GMP-bound
forms (Figs. 5A and 6A) shows that most NOEs are
identical between the two forms of GK. However, there are three pairs
of residues (Yb and Fi/Fj; Tyr-78 and Fi/Fj; Fc and Fd) with NOEs in
the free form but not in the GMP-bound form and another three pairs of residues (Ya and Fb; Tyr-50 and Fi/Fj; Fc and Fi/Fj) that show NOEs in
the GMP-bound form but not in the free form. In contrast, comparison of
the NOESY spectra of the WT GK and Y78F reveals that only a few NOEs
(one in the free form and four in the GMP-bound form) become very weak
or disappear in the mutant spectra. No additional NOE peaks appear in
the mutant spectra. The results suggest that there are conformational
changes upon binding of GMP. However, the conformations of the free
form and the GMP-bound form are very similar. This is characteristic of
the domain movements resulting from hinge and shear motions (34).
Paramagnetic probes have been used to aid identification of surface
residues by NMR (35-38). Addition of the paramagnetic probe TEMPOL
resulted in significant attenuation of many cross-peaks (Figs.
3B and 4B). However, it did not cause any
significant changes in their chemical shifts, indicating that TEMPOL
does not bind to GK. Furthermore, qualitatively, TEMPOL accessibility
determined for the GMP complex is consistent with the solvent
accessibility calculated from the crystal structure coordinate (24).
Thus the attenuation of the cross-peaks is likely due to the exposure of the side chains to TEMPOL but not specific binding. Binding of GMP
brings protection of several aromatic residues from the paramagnetic
bleaching, including Tyr-50 and Tyr-78. However, Fc becomes more
accessible to TEMPOL in the presence of GMP. As mentioned earlier, only
Tyr-50 and Tyr-78 are in close vicinity of the bound GMP. Thus the
changes in the TEMPOL accessibility upon binding of GMP are likely due
to substrate-induced changes in conformation and/or dynamics.
Müller et al. (30) have recently determined the
crystal structure of free E. coli adenylate kinase. Comparing it with the crystal structure of the same enzyme in complex
with Ap5A, a bisubstrate analogue, they have noticed
significant differences in the distribution of B-factors between the
two structures. On the basis of B-factors, they have suggested that the
lid and AMP-binding domains are mobile in the free form and become
immobilized upon binding of Ap5A. On the other hand, two
loops (4-
3 and
5-
4) are fixed in the free form and become
mobile upon binding of Ap5A. They have proposed that the
two mobile loops are the "energetic counterweight" that prevent the
enzyme from being trapped in an energetic well. It is of great interest
to see how substrate-induced conformational and dynamic changes in GK
correlate with catalysis.
In conclusion, we have shown that the hydrogen bond between hydroxyl group of Tyr-78 and the phosphate of GMP is critical for binding of GMP and catalysis. It stabilizes the GK·GMP complex by 1.7 kcal/mol, the ternary complex by 1.8 kcal/mol, and the transition state by 4.6 kcal/mol. Tyr-78 is not essential for proper folding of GK but it contributes to the conformational stability by 1.0 kcal/mol. In addition, we have demonstrated that GK undergoes conformational and dynamic changes upon binding of GMP.
We thank Dr. Genbin Shi for preparation of Fig. 1.