Monitoring Active Site Alterations upon Mutation of Yeast Pyruvate Kinase Using 205Tl+ NMR*

The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 ± 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p ≫ 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 ± 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 Å from 6.0 ± 0.2 Å observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.

The refined x-ray structures of the YPK-K ϩ -Mn 2ϩ -phosphoglycolate and YPK-K ϩ -Mn 2ϩ -phosphoglycolate-FBP complexes have been solved at 3 Å resolution (5). Phosphoglycolate is a structural analog of the substrate PEP that lacks the C-3 carbon. From the x-ray data, the active site Thr-298 is in the correct orientation to serve as the proton donor to the C-3 of the enolate of pyruvate, the enzyme-bound intermediate (Fig. 1). The role of Thr-298 in the YPK-catalyzed reaction has been addressed by mutation of this residue to serine, alanine (6), and to cysteine. 2 Studies of these mutants suggest that Thr-298 is not the proton donor to the enolate of pyruvate but that enzyme-bound water serves this function. Far-UV CD analysis indicates that none of the mutations cause any significant change in the secondary structure and that wild type and mutant YPK enzymes are folded into a similar, if not identical, structure. Physical and kinetic studies with the Thr-298 mutants of YPK indicate that the single amino acid mutations at the active site can trigger long range effects. These effects were observed at the FBP-binding site, located more than 40 Å away from the active site (5). The Thr-298 residue is about 6.5 Å from the enzyme-bound divalent cation and 8.9 Å from the monovalent cation at the active site ( Fig. 1) (5).
All three Thr-298 mutants of YPK, T298S, T298C and T298A, showed altered k cat and k cat /K m,PEP values and altered kinetic cooperativity with PEP relative to wild type PK (6). It is possible that these alterations in kinetic parameters of the YPK Thr-298 mutants may, in part, be explained by structural changes at the active site as a result of the single amino acid mutations. Such alterations may be too subtle to be monitored by methods such as CD and fluorescence spectroscopy, which are sensitive to more gross changes in protein structures.
When performing site-directed mutagenesis studies, the possibility of local structural alterations introduced upon mutation tends to be overlooked in the interpretation and assessment of overall properties and behavior of the resulting protein mutants relative to the wild type protein. The ability to address these questions is normally limited by lack of appropriate experimental tools. Yeast PK has an advantage in that it is amenable to such an analysis. Measurement of the internuclear interactions between the mono-and divalent cations, both located at the active site, provides a specific and sensitive method to monitor such structural alterations in YPK. In vitro, Tl ϩ has been shown to be a good substitute for K ϩ in wild type YPK (7).
Thallium is particularly well suited for NMR experiments because of its intrinsic nuclear properties. 205 Tl ϩ (70.5% natural abundance) is one of the two stable isotopes of thallium with a nuclear spin of 1 ⁄2. The relative receptivity (receptivity is sensitivity multiplied by natural abundance) of 205 Tl ϩ is 0.1355, in contrast to 39 K ϩ with a value of 0.000473. 1 H has an * This work was supported in part by Research Grant DK 17049 from the National Institutes of Health (to T. N.) and by the University of Notre Dame. 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  assigned value of 1 (8). This makes 205 Tl ϩ the fourth most receptive spin ϭ 1 ⁄2 nuclide and suggests that 205 Tl ϩ is a good spectroscopic replacement probe for potassium. The similarity of the crystalline ionic radii of Tl ϩ (1.44 Å) and K ϩ (1.37 Å) also suggests that thallous ion is a good replacement at the monovalent cation site. 205 Tl ϩ has been used previously to study ligand-induced conformational changes by NMR in muscle PK (9) and in YPK (7). Advantage was taken of the electron-nuclear interactions between 205 Tl ϩ and the unpaired electronic spin of Mn 2ϩ , which is a good substitute for Mg 2ϩ , the physiological divalent activator of these enzymes. The relaxation times of 205 Tl ϩ are strongly affected by this interaction, which in turn is very sensitive to the dipolar distance between the interacting nuclei. As a consequence, small changes in the distance between Tl ϩ and Mn 2ϩ at the active site of YPK that may be induced by single amino acid mutation will be reflected in amplified 205 Tl ϩ relaxation rate effects.
The goal of the present study is to monitor possible structural alterations introduced at the active site of YPK by mutation of Thr-298. The interaction of the monovalent cation with the T298S, T298C, and T298A mutants of YPK has been investigated by 205 Tl ϩ NMR and compared with the interaction with wild type YPK. The Tl ϩ -Mn 2ϩ distances at the active site of YPK have been calculated from the paramagnetic contribution of Mn 2ϩ to the longitudinal relaxation rates of 205 Tl ϩ bound to several complexes of the Thr-298 mutants and to wild type YPK. The primary results of these studies demonstrate that mutations of Thr-298 in YPK result in changes in the interactions between the monovalent cation and the divalent cation. These cations interact with the phosphoryl group of the substrate at the active site of YPK (5).

EXPERIMENTAL PROCEDURES
Materials-L-(ϩ)-Lactate dehydrogenase from rabbit muscle was purchased from Roche Applied Science. Wild type, T298S, T298C, and T298A yeast pyruvate kinases were constructed, expressed, and purified as described by Mesecar and Nowak (10). The cyclohexylammonium salts of PEP, ADP, and of FBP, disodium NADH, glycerol, and MES buffer were purchased from Sigma. Thallium nitrate was obtained from Aldrich. Tetramethylammonium hydroxide pentahydrate (TMA-OH) and tetramethylammonium nitrate (TMA-NO 3 ) were purchased from Acros Organics. Deuterium oxide (99.9%) was obtained from Cambridge Isotope Laboratories. The Bio-Gel® P-6 DG desalting gel was purchased from Bio-Rad.
Tl ϩ Activation Studies-YPK was assayed by following the decrease in absorbance at 340 nm due to NADH oxidation using the coupled assay with lactate dehydrogenase (11). The activity assays were performed at 22°C. The specific activity of YPK is expressed as mol of NADH oxidized per min/mg of protein. The concentration of YPK was determined by its absorbance at 280 nm. The extinction coefficient used for YPK is ⑀ 280 ϭ 0.51 (mg/ml) Ϫ1 cm Ϫ1 . For Tl ϩ activation studies with YPK, the assay conditions were as follows: a 1-ml assay mixture contained 100 mM MES (pH 6.2, adjusted with TMA-OH), 4% glycerol, TMA-NO 3 to 200 mM total salt concentration, 10% D 2 O, 4 mM Mn(NO 3 ) 2 , 5 mM PEP (cyclohexylammonium salt), 5 mM ADP (cyclohexylammonium salt), and 1 mM FBP (cyclohexylammonium salt), when present. Prior to Tl ϩ activation experiments, YPK was desalted on a Bio-Gel® P-6 DG column with a thin Chelex layer on top that was pre-equilibrated with 100 mM MES, pH 6.2 (adjusted with TMA-OH), 20% glycerol. Care was taken in these studies to eliminate activating monovalent cations such as K ϩ and Na ϩ . Thallous ion was added as TlNO 3 , and 10% D 2 O was included in the assay mixture to simulate NMR conditions. The total salt concentration was kept constant at 200 mM by the addition of non-activating TMA-NO 3 .
The initial velocity activation data were fit to either a noncompetitive substrate inhibition model (Equation 1) or to Equation 2, depending on which model best describes the experimental data. K a and K I values represent the activation constant and inhibition constant for Tl ϩ , respectively.
Tl ϩ NMR-NMR experiments were carried out on Varian 300 MHz (173.02 MHz) and Varian 600 MHz (346.04 MHz) Unity Plus instruments. Tuning of the probes to the thallium resonance was achieved by using a home-built inductor rod. The 90°pulse widths were calibrated prior to each experiment and typically ranged from 8 to 10 s at 300 MHz (173.02 MHz) and from 36 to 37 s at 600 MHz (346.04 MHz). D 2 O (10%) was included as an internal field-frequency lock. Samples with YPK were referenced to an external TlNO 3 sample that was identical in TlNO 3 concentration with the YPK sample and set at 0.0 ppm. At 300 MHz (173.02 MHz) the spectral width was 14,000 Hz, and 16,000 data points were used. The spectral width at 600 MHz (346.04 MHz) was 28,000 Hz, and 34,000 data points were used. The number of acquired transients ranged from 8 to 40. To improve signal to noise, a 2-40-Hz exponential line broadening function was used with the enzyme samples. In most of the cases, the line broadening was 2-9% of the measured line width. All spectra were acquired at 22 Ϯ 0.1°C.
Prior to the NMR experiments, YPK was desalted on a Bio-Gel® P-6 DG spin column with a thin Chelex layer (ϳ5 mm) on top that was pre-equilibrated with 100 mM MES, pH 6.2 (adjusted with TMA-OH), 20% glycerol. The typical concentration range of desalted YPK was 10 -15 mg/ml. Typical NMR samples contained in 0.7 ml: 100 mM MES, pH 6. ] T were corrected for dilution, although these corrections were less than 3% (the subscripts T and f denote total and free, respectively). The external TlNO 3 reference contained 100 mM MES, pH 6.2, 4% glycerol, 15-90 mM TlNO 3 , and TMA-NO 3 to 200 mM total salt concentration.
Longitudinal relaxation rates of 205 Tl ϩ (1/T 1 ) were determined using the inversion-recovery method (180°--90°acquire) (12). Typically 12-14 values were used and the data were fit to Equation 3, in which I is the peak height and A and B are constants. Deviations from a single exponential fit to the T 1 relaxation data were typically 2-6%.
Transverse relaxation rates (1/T 2 ) were determined from the line widths of the resonance signals according to Equation 4, which is based on the assumption of exponential relaxation. In Equation 4, ⌬1 ⁄2 is the value for the line width at half-peak height (1 ⁄2 ) corrected for the artificial line broadening (LB) according to Equation 5.
FIG. 1. Active site structure of yeast pyruvate in complex with K ؉ , Mn 2؉ , phosphoglycolate, and FBP (5). K ϩ is shown in gold, Mn 2ϩ in blue, and the bound substrate analogue phosphoglycolate (PG) is depicted with the phosphorous atom in purple and oxygen atoms in red. This view of the active site is obtained from the YPK structure published previously (5).
T 1 and T 2 values were determined at 5-6 concentrations of Mn 2ϩ . The paramagnetic contribution to the relaxation rates (1/T 1p and 1/T 2p ) were calculated from the slope of a plot of relaxation rate against concentration of Mn 2ϩ . The values of 1/T 1p and 1/T 2p were normalized by the factor p ϭ [Mn 2ϩ ] b /[Tl ϩ ] T , as described by Mildvan and Cohn (13), to generate values of 1/pT 1p . Under conditions of fast exchange 1/pT 1p ϭ 1/T 1M . K D Determination-Binding of Mn 2ϩ to the YPK-Tl ϩ -PEP-FBP complexes of WT, T298S, and T298A YPK enzymes was determined by loss of EPR signal of free Mn 2ϩ upon binding. All data were fit to a hyperbolic binding model. Measurements were performed using a Bruker ER 100E electron spin resonance spectrometer. The experimental conditions used are as follows: frequency 9.88 GHz, modulation amplitude 1.6 ϫ 10 gauss peak-to-peak, time constant 500 ms, field set at 3450 G, sweep time 200 -500 s, and variable gain.
For all YPK-Tl ϩ -PEP complexes and for the T298C-Tl ϩ -PEP-FBP complex, the binding constant for Mn 2ϩ was determined by tryptophan fluorescence titrations. Measurements were performed on an SLM-Aminco 8100 spectrofluorimeter. Fluorescence titrations were performed by monitoring the change in fluorescence intensity at 334 nm, with excitation at 295 nm upon titration with Mn 2ϩ . Data were fit to either a hyperbolic or a cooperative model, depending on which one best describes the experimental data. The enzyme concentration was 0.1-0.2 mg/ml, and the buffer and ligand concentrations were the same as in the NMR experiments. Mn 2ϩ binding to the T298C-Tl ϩ -PEP-FBP complex could not be determined directly by fluorescence titration since no additional quenching of tryptophan was observed upon ligand binding to FBP complexes of YPK. Instead the thermodynamic box depicted in Scheme 1 was used to determine the dissociation constant for Mn 2ϩ binding to T298C-Tl ϩ -PEP-FBP. This method requires intrinsic dissociation constants 3 (KЈ D ), which were calculated from the respective measured dissociation constants (K D ) and using the model for cooperativity in binding for YPK developed by Bollenbach (14) based on Pauling's treatment for hemoglobin (15). In Scheme 1, EЈ is T298C-Tl ϩ -PEP, M is Mn 2ϩ , and A is FBP. KЈ 4 was calculated from the relationship Correlation Time ( C ) and Correlation Function (f( C )) for Mn 2ϩ -Tl ϩ Interaction-Applications of the longitudinal (1/T 1 ) and transverse (1/T 2 ) relaxation rates of a nucleus bound in the vicinity of a paramagnetic probe to study enzyme-metal complexes have been reviewed previously (13,16,17). It has been demonstrated that the longitudinal relaxation rate of magnetic nuclei of a complexed ligand (1/T 1M ) can be used to calculate the dipolar distance (r) between these nuclei (e.g. Tl ϩ ) and the paramagnetic metal ion (e.g. Mn 2ϩ ) (18,19). In the enzyme complexes that have been studied, the paramagnetic contribution by a metal such as Mn 2ϩ to the 1/T 1 relaxation rates of Tl ϩ , considering only dipolar relaxation, is given by the simplified Solomon-Bloembergen equation (Equation 6), where S is the electron spin quantum number; ␥ I is the nuclear gyromagnetic ratio; g is the electronic "g" factor; ␤ is the Bohr magneton; c is the correlation time for the Mn 2ϩ -Tl ϩ interaction; and I and S are the nuclear and electron resonance frequencies, respectively. In Equation 6 the term due to the electron-nuclear hyperfine interaction has been omitted since it appears to be negligible for most ligands that interact with Mn 2ϩ . In macromolecular complexes in which large enhancements of 1/T 1p are observed, S 2 c 2 becomes Ͼ Ͼ1 and the term 7 c /(1 ϩ S 2 c 2 ) in Equation 6 is negligible. Hence, the correlation function, f( c ), for the dipolar term of the Solomon-Bloembergen equation (Equation 6) is simplified to Equation 7, The correlation time c was determined from the frequency dependence of 1/T 1M at I,1 (173.02 MHz) and I,2 (346.04 MHz) and by assuming that c is frequency-independent. Knowing c , the correlation function f( c ) can be calculated from Equation 7. Once f( c ) is determined, the Tl ϩ -Mn 2ϩ distance r, in Angstroms, can be calculated, according to

RESULTS
Tl ϩ Activation and Mn 2ϩ Binding-Tl ϩ activation studies were performed for wild type YPK and the Thr-298 mutants with Mn 2ϩ as the divalent activator, and in the absence and in the presence of FBP, respectively (Fig. 2, A-C). Wild type YPK and the Thr-298 mutant enzymes show no measurable YPK activity in the absence of an activating monovalent cation (K ϩ or Tl ϩ ) with TMA or cyclohexylammonium ions present as counter cations (Ͻ0.1 units/mg, the sensitivity limit of the assay). This is in agreement with the previous observations with muscle PK (20) and with wild type yeast PK (2, 7) that these enzymes have an absolute requirement for an activating monovalent cation.
In the presence of the heterotropic activator FBP, Tl ϩ can activate wild type YPK to 85% the activity in the presence of K ϩ (Table I). With T298S, T298C, and T298A, Tl ϩ is a 1.2-1.8fold better activator than is K ϩ based on the measured k cat values. With Mn 2ϩ as the divalent activator, Tl ϩ has a steadystate activator constant (K a ) that is ϳ35-fold smaller than the value for K ϩ with wild type and with T298A YPK. The K a for Tl ϩ compared with K ϩ is ϳ11-fold and 17-fold smaller with T298S and T298C YPK, respectively. At higher Tl ϩ concentrations, inhibition is observed with wild type YPK (data not shown) and with the mutants T298S ( Fig. 2A) and T298C (Fig.  2B). With both mutants, the inhibition constant for Tl ϩ , K I , is increased 3-4-fold relative to the value with wild type YPK. These values are summarized in Table I, along with the results of activation studies with K ϩ . T298A shows no inhibition by Tl ϩ in the concentration range studied (Fig. 2C).
Activation by monovalent cations shows no kinetic cooperativity in the absence or presence of FBP. In the absence of FBP, Tl ϩ activates wild type YPK and the mutants T298S and T298C with k cat values similar to the values measured in the presence of FBP (Table I). With T298A, Tl ϩ activation in the presence of FBP gives a 32% increase in k cat . The values measured for the Tl ϩ activation constant, K a , and the inhibition constant, K I , with wild type and T298S YPK, and for K a with T298A YPK are similar in the absence and in the presence of FBP. With T298C, the presence of FBP decreases the apparent activation constant for Tl ϩ (K a ) and increases the inhibition constant for Tl ϩ (K I ) both by a factor of 3. No inhibition of T298A is observed with Tl ϩ in the absence or presence of FBP (Fig. 2C).
The mutation of Thr-298 to serine, cysteine, and to alanine results in decreased k cat values upon activation by Tl ϩ and by K ϩ . Increased values for the kinetic constants K a (Tl ϩ ), K a (K ϩ ), and K I (Tl ϩ ) relative to those of wild type YPK are also measured (Table I).
The following Tl(NO 3 ) concentrations were used for the NMR experiments with YPK based on the data in Table I: 15  and 70 and 90 mM for T298A. With the exception of T298C in the absence of FBP, these concentrations were 10 -15-fold greater than the K a for Tl ϩ to ensure saturation and 7-10-fold less than its K I value (with wild type and T298S) to minimize the effects from inhibition. In the case of T298C in the absence of FBP, the large K a for Tl ϩ limited the Tl(NO 3 ) concentration used in the NMR experiments to 60 mM, which is only 3-fold greater than K a and 2.5-fold less than K I .
Mn 2ϩ Binding Studies-To determine the contribution of Mn 2ϩ to the 205 Tl ϩ relaxation rates of enzyme-bound thallium, the amount of Mn 2ϩ bound to YPK must be known. The dissociation constants for Mn 2ϩ binding to the complexes of wild type YPK and the Thr-298 mutant enzymes were measured and are summarized in Table II. The dissociation constants for Mn 2ϩ binding to the YPK-Tl ϩ -PEP complexes measured with wild type and with T298S and T298C mutants are similar. Mn 2ϩ binding to the T298A-Tl ϩ -PEP complex is 20 times weaker than Mn 2ϩ binding to the similar wild type complex. Table II shows that a 5-fold increase of Tl ϩ concentration in the T298S-Tl ϩ -PEP complex or a 6-fold increase of Tl ϩ concentration in the WT-Tl ϩ -PEP-FBP complex results in no significant effect on K D,Mn 2ϩ. This suggests that Tl ϩ does not significantly compete for the Mn 2ϩ -binding site. The presence of the heterotropic activator FBP tightens the binding of Mn 2ϩ to each of the enzyme complexes. The measured K D,Mn 2ϩ values for the YPK-Tl ϩ -PEP-FBP complexes are similar for wild type and mutant enzymes. The K D for Mn 2ϩ binding to the T298C-Tl ϩ (60 mM)-PEP-FBP complex (KЈ 4 ) was calculated from the relationship KЈ 4 ϭ KЈ 1 KЈ 3 /KЈ 2 , as described under "Experimental Procedures." The values for KЈ 1 , KЈ 2 , and KЈ 3 are 39, 986, and 57.5 M, respectively.
205 Tl ϩ NMR Studies-Depending on the YPK-Tl ϩ complex studied, solutions of 15, 60, 70, and 90 mM Tl(NO 3 ) were used as reference in the NMR experiments. In each case, the thallium resonance was set to 0.0 ppm. The reference solution contained the same concentrations of Tl(NO 3 ), buffer, and total salt as the samples containing enzyme. All 90°pulse width calibrations were performed on the respective reference solutions. The measured line width of thallium in solution was 4 at 173 Hz and 5 at 346 Hz. Addition of Mn(NO 3 ) 2 , up to 1 mM, to a solution of 70 mM Tl(NO 3 ) in buffer had no effect on the T 1 or T 2 values (the highest concentration of total Mn(NO 3 ) 2 used in the NMR experiments was 0.7 mM). This is in agreement with the previous observation of Loria and Nowak (7) where the addition of up to 5 mM Mn(NO 3 ) 2 to a solution of 15 mM Tl(NO 3 ) in buffer caused no T 1 or T 2 effects. Fig. 3, A-D, shows the comparative NMR spectra of Tl(NO 3 ) in solution and in the presence of wild type YPK and the Thr-298 mutants as their enzyme ϩ PEP and enzyme ϩ PEP ϩ FBP complexes, at 173 MHz. At saturating levels of PEP, the thallium resonance is broadened to 56 Hz in the presence of 78 M wild type YPK sites (Fig. 3A-2), to 43 Hz in the presence of 80 M T298S sites (Fig. 3B-2), to 56 Hz in the presence of 134 M T298C sites (Fig.  3C-2), and to 41 Hz in the presence of 101 M T298A sites (Fig.  3D-2). The respective downfield chemical shifts for the thallium resonance in the YPK-Tl ϩ -PEP complexes were 16.5 ppm (wild type), 14.3 ppm (T298S), 14.8 ppm (T298C), and 14.5 ppm (T298A). Each enzyme elicits different downfield shifts even when normalized for the concentration of enzyme. This suggests that enzyme-bound Tl ϩ "sees" different environments in wild type YPK and in each of the Thr-298 mutants. In the YPK-Tl ϩ -PEP-FBP complex of each enzyme species, there was a slight change in the line broadening and in the chemical shift of the thallium resonance relative to values from the YPK-Tl ϩ -PEP complex (Fig. 3, A-3 to D-3). The addition of Mn 2ϩ to each of the YPK-Tl ϩ (ϩPEP, ϮFBP) complexes results in an increase in the 1/T 1 and 1/T 2 values for thallium, and the increase is proportional to the Mn 2ϩ concentration (data not shown). For each complex, there is a slight upfield chemical shift (Ͻ0.5 ppm) of the thallium resonance upon addition of Mn 2ϩ (data not shown). It was demonstrated previously (7) that the addition of Mg 2ϩ to Tl ϩ complexes of wild type YPK . The fits generated the curves through the data. has no effect on the 1/T 1 and 1/T 2 values nor on the chemical shift for thallium. This suggests that the observed effect of the divalent metal on the Tl ϩ relaxation rates is due to the paramagnetic properties of Mn 2ϩ .
The chemical shifts and paramagnetic effects on relaxation at 346 MHz were similar to those measured at 173 MHz for each of the YPK-Tl ϩ complexes of wild type YPK and of the T298S mutant.
A summary of the normalized paramagnetic contributions (1/pT 1p and 1/pT 2p ) to the 1/T 1 and 1/T 2 values measured at 173 and at 346 MHz is presented in Table III. For all YPK-Tl ϩ complexes studied, 1/pT 2p Ͼ Ͼ 1/pT 1p . As argued previously (21), 1/pT 1p approximates 1/T 1M (see below), the relaxation rate of the nucleus bound to the macromolecule. The data are listed as such in Table III. In the WT-Tl ϩ (15 mM)-PEP complex at 173 MHz, the 1/T 1M value is 2350 Ϯ 300 s Ϫ1 (Table III), in good agreement with the value of 2540 Ϯ 1200 s Ϫ1 obtained by Loria and Nowak (7) under the same conditions. The addition of the allosteric activator FBP to this complex does not result in a further change in 1/T 1M , as also observed previously (7). An increase in the Tl ϩ concentration by a factor of 6 in the WT-  (Table III). The addition of FBP to the enzyme-Tl ϩ -PEP complex of the Thr-298 mutants results in a small but significant change in 1/T 1M . In the enzyme-Tl ϩ -PEP-FBP complex of the Thr-298 mutants, the values for 1/T 1M differ among these mutants and are 2.5-4-fold larger than the 1/T 1M value in the wild type-Tl ϩ (15 mM)-PEP-FBP complex.
The values for 1/T 1M are normalized for 100% saturation (activation) with Tl ϩ , 1/T 1M,100 (see below). These values are also summarized in Table III. The 1/pT 2p values measured at 173 MHz for the Tl ϩ complexes of wild type and of Thr-298 mutants are on the order of 10 5 -10 6 s Ϫ1 . These values are 2-3 orders of magnitude larger than the 1/T 1M values for these complexes (Table III).
The measurement of 1/T 1M values at 346 MHz for wild type YPK and the T298S mutant enzyme-Tl ϩ -PEP and enzyme-Tl ϩ -PEP-FBP complexes indicates that there is a frequency dependence for 1/T 1M , based on Equation 6 and that the measured values reflect relaxation (Table III). There is no frequency dependence for the 1/pT 2p values for these complexes (Table III).
Calculation of 1/T 1M Normalized for 100% Saturation with Tl ϩ -Each of the enzyme complexes studied has a different activator constant (K a ) and inhibitor constant (K I ) for Tl ϩ , thus requiring different concentrations of Tl ϩ to optimize complex formation (Table I). For a more accurate comparison of the 1/T 1 relaxation rates measured for the YPK-Tl ϩ complexes at different concentrations of Tl ϩ , hence at different saturation levels with Tl ϩ , the relaxation rates were normalized to 100% saturation (1/T 1M,100 ). For each enzyme-Tl ϩ complex, the value for 1/T 1M,100 was calculated as 1/T 1M,100 ϭ ((1/T 1M )/(%A)) ϫ 100, where 1/T 1M is the calculated and normalized (by 1/p) longitudinal relaxation rate of 205 Tl ϩ in the enzyme-Tl ϩ complexes studied, and %A is the percent saturation (activation) with Tl ϩ . The percent saturation (activation) with Tl ϩ was calculated from Equation 2, as ( 0 /V) ϫ 100 ϭ {[Tl ϩ ]/ (K a ϩ[Tl ϩ ])} ϫ 100, assuming no inhibitory effect at higher Tl ϩ concentrations (Table III).
The logic to normalize the 1/T 1M values for 100% saturation with Tl ϩ is described. The activation of the YPK complexes by Tl ϩ has an inhibitory component at higher Tl ϩ concentrations (see Fig. 2). The measured 1/T 1 values for 205 Tl ϩ in several of   these complexes consequently have an inhibitory component. Since the mechanism of inhibition is unclear, we minimized the effect of the inhibitory component on the 1/T 1 values of Tl ϩ for the YPK-Tl ϩ complexes studied. Presumably Tl ϩ binds at two sites on the enzyme, an activator site and an inhibitor site, and these sites are independent. Each of these sites has a contribution to the overall observed 1/T 1M . This model is described by Equation 9.  -1, B-1, C-1, and D-1, 1 Hz; A-2 and A-3, 10 Hz; B-2, B-3, D-2, and D-3, 3 Hz; and C-2 and C-3, 5 Hz. ent. The normalization of 1/T 1M for 100% saturation (activation) with Tl ϩ can thus be applied to the enzyme-Tl ϩ complexes.
The model described by Equation 9 was tested with the T298S-Tl ϩ -PEP complex for 15, 35, and 70 mM Tl ϩ , respectively, at 346 MHz by using the above-described approach. The result was generalized for wild type and the three Thr-298 mutant enzymes, assuming that wild type and mutant en-  (Table III). The (1/T 1M ) A value is therefore used for comparing 1/T 1M values in enzyme-Tl ϩ complexes of wild type YPK and the Thr-298 mutants. For simplicity, the 1/T 1M,100 values will be used for analysis in this study. In the case of the T298S-Tl ϩ -PEP complex at 346 MHz, the term (%A/[E] T )(1/T 1M ) A shows a saturation dependence as a function of Tl ϩ concentration (Fig. 4). A fit of this dependence to Equation 2 gives K a ϭ 13 Ϯ 3 mM and the value of 5000 Ϯ 300 s Ϫ1 for 1/T 1M at saturating Tl ϩ concentration. These values are in reasonable agreement with K a ϭ 6.0 Ϯ 0.5 mM (Table I) measured kinetically and to 1/T 1M,100 ϭ 5670 Ϯ 280 s Ϫ1 calculated for the T298S-Tl ϩ (70 mM)-PEP complex. DISCUSSION Recent studies (6) with YPK were performed to determine the functional group at the active site that is responsible for the enzyme-catalyzed protonation of the enolate of pyruvate. In the yeast enzyme, the 100% conserved Thr-298 is in the optimal location to perform this function (5). Several mutations at Thr-298 were constructed, and the mutant proteins were isolated and characterized. Each mutant enzyme has different kinetic characteristics. The results led to the conclusion that a molecule of bound water at the active site serves as the proton donor (6). In an effort to characterize further the effect of these mutations at the active site, the interaction between the enzyme-bound divalent activator (Mn 2ϩ ) and the monovalent activator (Tl ϩ ) was investigated in each of the mutants by appropriate NMR studies.
Thallium Activation-Wild type YPK is activated by Tl ϩ with Mn 2ϩ as divalent activator and in the presence of FBP with a k cat value similar to that measured with K ϩ (Table  I). With the three Thr-298 mutants, Tl ϩ is a better activator than is K ϩ based on k cat values. It has been suggested by Loria and Nowak (7) that the monovalent cation in PK may orient either an enzymic group for substrate binding and/or catalysis or the phosphate group for attack by the incoming nucleophile. It is possible that the greater k cat value with Tl ϩ than with K ϩ for the Thr-298 mutants is due to a more favorable accommodation of Tl ϩ at the active site with respect to the group whose orientation is aided by the monovalent cation. Tl ϩ binds with higher affinity than the physiological activator K ϩ to wild type YPK and Thr-298 mutants (Table I), as observed with other enzymes that require a monovalent cation such as Na ϩ or K ϩ (22). The ionic radii of thallium and potassium are 1.44 and 1.37 Å, respectively. These results and previous studies by Raushel and Villafranca (23), Markham (22), and Loria and Nowak (7) suggest that size has an important role in monovalent cation activation of enzymes that require such a cofactor for activity. Binding of Tl ϩ to YPK is affected by mutation of Thr-298 in the active site. The apparent K a values for Tl ϩ with the Thr-298 mutant enzymes are five to eight times greater than the values measured with wild type YPK. Mn 2ϩ -activated T298C in the absence of FBP is an exception; the value for K a for Tl ϩ is 20-fold larger than the value determined with wild type YPK. At higher Tl ϩ concentrations, wild type YPK, T298S, and T298C show inhibition of activity (Fig. 2, A and B). No inhibition by Tl ϩ is observed with T298A (Fig. 2C). Perhaps with . The best fit parameters are K a ϭ 13 Ϯ 3 mM, the activation constant for Tl ϩ , and 1/T 1M,100 ϭ 5000 Ϯ 300 s Ϫ1 , the value for 1/T 1M at saturating Tl ϩ concentration at the activator site.

TABLE III
Normalized longitudinal and transverse relaxation rates for the T1 ϩ -Mn 2ϩ interaction in wild type and Thr-298 mutant YPK complexes Normalized longitudinal and transverse relaxation rates were determined for YPK complexes of wild type and Thr-298 mutants. Experiments were performed at 173 MHz and 346 MHz as described under "Experimental Procedures." T298A, the K I for Tl ϩ is much greater. It was suggested previously (7) that the inhibition observed is due to Tl ϩ binding at the divalent metal site at higher Tl ϩ concentrations. Binding of Mn 2ϩ to several of the enzyme-Tl ϩ complexes (Table II) at higher Tl ϩ concentration shows no increase in K D,Mn 2ϩ, which argues against the above explanation. The changes in Tl ϩ binding to the Thr-298 mutants of YPK relative to wild type YPK may be due to different ligand-induced conformational changes at the active site of the mutant enzymes than those induced in the wild type enzyme. With T298C, the presence of FBP decreases the K a for Tl ϩ and increases the K I for Tl ϩ , both by a factor of 3. Fluorescence titration experiments with T298C YPK show that the K D for FBP is 2-fold greater to the T298C-Tl ϩ -PEP complex than to the T298C-K ϩ -PEP complex and 4 times greater to the T298C-Tl ϩ -Mn 2ϩ -PEP complex than to its K ϩ counterpart. 2 The concentration of PEP (5 mM) was saturating under kinetic conditions for wild type YPK and the Thr-298 mutants. The K m,PEP value was measured by steady-state kinetics for all enzymes studied in the presence of saturating Tl ϩ concentrations (15 mM for wild type, 90 mM for T298S and T298A, and 60 mM for T298C), at 200 mM total salt concentration, and in the absence or in the presence of FBP (data not shown). In all cases, the values for K m,PEP were in the micromolar range (10 -500 M), similar to the values measured in the presence of 200 mM K ϩ . These results demonstrate that altered K a and K I values for Tl ϩ are not due to subsaturating concentrations of PEP with the Thr-298 mutants.
The dissociation constants for Mn 2ϩ binding to the complexes of wild type YPK and of the Thr-298 mutants were determined (Table II). Mn 2ϩ binds to the enzyme-Tl ϩ -PEP complex for the wild type, T298S, and T298C enzymes with the same affinity. The T298A mutation weakens the binding of Mn 2ϩ to the enzyme-Tl ϩ -PEP complex by about an order of magnitude. With the T298S-Tl ϩ -PEP complex, a 5-fold increase of Tl ϩ concentration has no significant effect on K D,Mn 2ϩ. Table  III shows an increase in the observed 1/T 1M with increasing Tl ϩ concentration in the T298S-Tl ϩ -PEP complex. If Tl ϩ were binding at the Mn 2ϩ site at higher concentrations, a decrease of the paramagnetic effect of Mn 2ϩ on 205 Tl ϩ relaxation would be observed due to Mn 2ϩ displacement by Tl ϩ . The increase in 1/T 1M at higher concentrations of Tl ϩ may result from greater occupancy at the inhibitory site of Tl ϩ . Similar results were obtained with rabbit muscle PK in the enzyme-Tl ϩ -PEP complex at 15 mM Tl ϩ and 100 mM Tl ϩ (7). These results indicate that Tl ϩ inhibition is not due to displacement of Mn 2ϩ and that the inhibitory site is near or at the active site of YPK. The presence of the heterotropic activator FBP tightens the binding of Mn 2ϩ to YPK, and the measured K D,Mn 2ϩ values are similar for the wild type and mutant complexes (Table II). With WT-Tl ϩ -PEP-FBP, a 6-fold increase of Tl ϩ concentration does not affect the K D,Mn 2ϩ value. The explanation for this observation may be the same as for the T298S-Tl ϩ -PEP complex (see above).
Longitudinal and Transverse Relaxation Rates-The NMR spectra for 205 Tl ϩ were measured in the presence of wild type YPK and the three mutants. The 205 Tl ϩ resonance in the PEP complex of wild type YPK is 2 ppm downfield of the 205 Tl ϩ resonance in the PEP complex of the Thr-298 mutants (Fig. 2,  A-2 to D-2). These data indicate different environments for the thallous ion in each of these complexes. Nuclear relaxation effects of bound Mn 2ϩ on the 205 Tl ϩ resonance were used to further investigate the nature of the environmental differences. Table III shows that for each complex studied, the normalized values of 1/T 2p are significantly larger than the normalized values of 1/T 1p . The measured and normalized value of 1/T 1p is frequency-dependent (Table III). Both observations suggest that the relaxation phenomenon and not chemical exchange is measured for 1/T 1p .
1/pT 2p determined for the enzyme-Tl ϩ -PEP and enzyme-Tl ϩ -PEP-FBP complexes of wild type YPK and T298S are independent of frequency (Table III). There are two possible explanations for this observation. Equation 10 describes the relationship between the measured relaxation rate (1/T 2p ), the residence time ( M ), the stoichiometry q, assumed to be 1, and the relaxation time (T 2M ) of bound Tl ϩ .
If only dipolar relaxation is considered, 1/T 2M is given by the simplified Solomon-Bloembergen equation (Equation 11). The more general form of Equation 11 has an additional scalar term for the relaxation rate (19). If scalar effects contribute to relaxation, then 1/T 2M is even greater. In macromolecular complexes in which large enhancements are observed, S 2 c 2 Ͼ Ͼ 1 and the correlation function of the dipolar term in Equation 11 is simplified to 4 c ϩ 3 c /(1 ϩ I 2 c 2 ). 1/pT 2p could be frequency independent if (a) M dominates the relaxation rate 1/T 2p in Equation 10 or (b) if in the simplified form of Equation 11, the frequency independent term 4 is much larger than the frequency dependent term, . If a is the case then 1/pT 2p ϭ 1/ M , the rate of chemical exchange. The temperature dependence data of 1/T 2p for enzyme-bound Tl ϩ in complexes of wild type YPK gave Arrhenius plots (1/T 2p versus 1/temperature) that had zero or negative slopes, depending on the complex (7). The frequency-independent value calculated for c is 0.65 ϫ 10 Ϫ9 s (see below). This value for 1/T 2M was calculated for the WT-Tl ϩ -PEP complex at both frequencies from the simplified form of Equation 11 5 assuming only dipolar effects and was compared with the measured 1/pT 2p values listed in Table III. This calculation eliminates argument b, suggests scalar contributions to 1/T 2M , and further indicates that chemical exchange contributes to 1/T 2p . Scalar superhyperfine coupling has been reported between VO 2ϩ (a Mn 2ϩ 4 It is assumed that c itself is not a function of frequency in this range of magnetic fields. 5 Fast exchange conditions were considered for 1/T 1p ; therefore 1/pT 1p ϭ 1/T 1M . The term S(S ϩ1)␥ 2 I g 2 ␤ 2 /15r 6 was calculated from the simplified form of Equation 6 using the values for 1/T 1M listed in Table  III.  Table III. analog) and 205,203 Tl ϩ bound at the active site of muscle PK (24). Reuben and Kayne (9) have also suggested scalar effects in the Mn 2ϩ -Tl ϩ interaction with muscle PK. The analysis indicates that transverse relaxation rates are not solely governed by dipolar interactions and therefore will not be used to calculate the Tl ϩ -Mn 2ϩ distance in the complexes of wild type YPK and the Thr-298 mutants that were studied.
The values of 1/pT 1p appear to be in fast exchange (Table III). From the Swift-Connick relationship for 1/pT 1p and 1/pT 2p (Equation 10), 1/pT 1p ϭ 1/T 1M . The existence of fast exchange conditions for 1/T 1p is also supported by the positive slopes in the Arrhenius plots for longitudinal relaxation rates of enzymebound Tl ϩ in complexes of wild type YPK, as reported by Loria and Nowak (7), and by the frequency dependence of 1/pT 1p (Table III).
Studies with muscle PK (7) indicate that c is not a function of frequency over the range of the magnetic field studied. Assuming similar properties of c with YPK, the correlation time for the Tl ϩ -Mn 2ϩ interaction in YPK was calculated using Equation 6 for values of 1/T 1M at 173 and 346 MHz. By using this method for the WT-Tl ϩ (15 mM)-PEP, WT-Tl ϩ (15 mM)-PEP-FBP, T298S-Tl ϩ (70 mM)-PEP, and T298S-Tl ϩ (90 mM)-PEP-FBP complexes, the following values for c were calculated: 0.77 Ϯ 0.52, 0.52 Ϯ 0.46, 0.70 Ϯ 0.31, and 0.54 Ϯ 0.36 ns, respectively. Although there is some variation in the c values estimated, these variations are probably due to experimental errors and limitations of the method. Values of c ranging from 0.52 to 0.77 ns result in a calculated distance range differing by less than 0.14 Å. For simplicity, it is assumed that the c values for each complex of wild type YPK and Thr-298 mutants are the same, and an average value of 0.65 Ϯ 0.35 ns will be used for further calculations. A similar approach has been previously used for estimation of correlation times for muscle PK complexes (7,23) and yeast PK complexes (7). These results indicate that the variations in 1/T 1M between wild type YPK and the various Thr-298 mutants are due to variations in the dipolar distances and not in c values.
Tl ϩ -Mn 2ϩ Distances in YPK Complexes-The Tl ϩ -Mn 2ϩ distances in the enzyme-Tl ϩ -Mn 2ϩ complexes of wild type YPK and Thr-298 mutants were calculated using Equation 8, in which the values for T 1M were normalized for saturation (T 1M,100 ) and are listed in Table III. The calculated Tl ϩ -Mn 2ϩ distances in complexes of wild type and the Thr-298 mutants of YPK are summarized in Table IV. For the WT-Tl ϩ (15 mM)-PEP complex the calculated inter-metal distance is 6.0 Ϯ 0.1 Å (Table IV). This value is in excellent agreement with the value of 6.1 Ϯ 0.3 Å determined by Loria and Nowak (7) and with the K ϩ -Mn 2ϩ distance of 5.8 Å measured by x-ray diffraction methods for the YPK-Mn 2ϩ -phosphoglycolate complex (5). The cal-culated Tl ϩ -Mn 2ϩ distance for the enzyme-Tl ϩ -PEP complex of the Thr-298 mutants is decreased by about 1 Å relative to wild type YPK.
The binding of the allosteric activator FBP to the enzyme-Tl ϩ -PEP complex does not induce any further change in the Tl ϩ -Mn 2ϩ distance in wild type YPK and Thr-298 mutants of YPK (Table IV). FBP binding to the enzyme-K ϩ -PEP complex of wild type YPK and Thr-298 mutants causes a large quenching in the tryptophan fluorescence (6) that is indicative of structural changes in the protein. It appears that binding of FBP to the enzyme-PEP complex induces global conformational changes but no additional local changes at the active site of YPK where the Tl ϩ and Mn 2ϩ ions bind. The values of 6.1 and 5.8 Å for the distance between the Mn 2ϩ and K ϩ in the YPK-Mn 2ϩ -phosphoglycolate-FBP and the YPK-Mn 2ϩ -phosphoglycolate complexes, respectively, are the same within the 3-Å resolution of the x-ray refinements of the two structures (5).
Ligand Exchange Kinetics-In addition to its applicability to yield structural information of enzyme-substrate complexes in solution, nuclear relaxation results may provide valuable kinetic information. The rates of chemical exchange of ligands into the environment of a paramagnetic center can be measured, depending upon the relative rates of chemical exchange and the various relaxation processes (Equation 10). The application of NMR to ligand exchange processes has been reviewed (25).
The chemical exchange seems to have an important contribution to the transverse relaxation rates of 205 Tl ϩ measured in all YPK-Tl ϩ complexes studied (see above). This allows the use of 1/pT 2p of 205 Tl ϩ as a lower limit of its exchange rate (k off ). The rate constants for formation of the YPK-Tl ϩ complexes with wild type and Thr-298 mutants (k on ) were calculated from the transverse relaxation rates 1/pT 2p ϭ 1/ M ϭ k off and the measured activator constants (K a ) listed in Table I. K a is treated as a dissociation constant (Table V). All calculated values for k on are on the order of 10 8 M Ϫ1 s Ϫ1 , suggesting diffusion-controlled binding of Tl ϩ to YPK complexes. These results are expected if chemical exchange rather than relaxation phenomenon is measured by 1/T 2p . The larger values for 1/pT 2p for Tl ϩ in the complexes with the mutant enzymes suggest faster exchange rates for Tl ϩ .
In conclusion, the results obtained with wild type YPK and the Thr-298 mutants indicate conformational alterations of the substrate and the cations at the active site of YPK upon mutation of Thr-298. Possible conformational alterations at the active site of YPK upon mutation of Thr-298 were suggested by the altered dissociation constants measured for PEP and Mn 2ϩ binding to various complexes of T298S, T298C, and T298A enzymes (6). The results presented in this study demonstrate TABLE V Rate constants k on for Formation of T1 ϩ Complexes of YPK Rate constants for formation of T1 ϩ complexes of YPK, k on , were calculated from K a ϭ k off /k on . The activation constant for T1 ϩ , K a , was assumed to be an equilibrium constant. The rate constant for dissociation of T1 ϩ , k off , was estimated from the magnitude of 1/pT 2p assuming that 1/pT 2p ϭ 1/ M , where 1/ M is the rate constant for chemical exchange (see "Discussion").
that mutation of the catalytically important Thr-298 residue induces a closer proximity of the monovalent and divalent cations that are located at the site of phosphoryl transfer (Fig.  1). Since both cations interact with the phosphate group of PEP (see Ref. 5), the alterations measured by 205 Tl ϩ NMR may reflect a conformational change of PEP at the active site. The substrate-induced conformational changes in YPK measured by 205 Tl ϩ NMR may, in part, explain the alteration in k cat and k cat /K m,PEP observed with T298S, T298C, and T298A (6). These results emphasize that interpretation and assessment of overall properties and behavior of mutant enzymes relative to the wild type protein must be done with caution. Amino acid mutation in a protein may introduce subtle structural alterations at the site of mutation or at remote sites. These structural changes may be, in part, responsible for the altered catalytic properties of the mutant enzymes. The unique properties of YPK have allowed for such detailed analyses.