Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.


13C NMR and Aspartate
Transcarbamylase 5977 catalysis by compressing the amino group of aspartate and the carbonyl group of carbamyl-P together in the active site (1,3). Studies by Beard and Schmidt (4) of the binding of succinate to the subunit.carbamyl-P complex by 'H NMR indicate that between pH 7 and pH 10, two protonated groups are involved in the conformational change. One, with pK about 8, participates directly in the binding of succinate and another, with pK about 7, is thought to be at a site distinct from the succinate binding site. The latter group may interact with the carbonyl group of carbamyl-P, and may be involved in the conformational change.
There are several recent reports in which 13C NMR of carbonyl carbon atoms has been used to provide information about enzyme mechanisms. These include the binding of bicarbonate to the proteins ribulose 1,5-diphosphate carboxylase (5), carbonic anhydrase (6), myoglobin (7), and transferrin (8). 13C NMR provides the possibility to examine a crucial detail of the catalytic mechanism of aspartate transcarbamylase, the postulated protonation of the carbonyl group of carbamyl-P, using this substrate directly rather than an analog which might interact with the enzyme somewhat differently.
The 13C chemical shift of carbonyl carbon atoms is determined largely by the local paramagnetic term in the Karplus and Pople theory (9,lO). This theory has been used successfully to interpret 13C NMR studies with model compounds: and we use it now to interpret the effects observed with aspartate transcarbamylase.
With model compounds, the dependence upon solvent indicates that 13C chemical shifts for carbonyl carbon atoms are relatively intensitive to polarity and highly sensitive to the presence of proton donors (ll).' In fact, there is a direct relationship between the solvent-induced downfield change in chemical shift of a carbonyl carbon atom and the ability of the solvent to form hydrogen bonds (13). With the usual assumptions of the Karplus and Pople theory, i.e. no change is sigma bonds, constant average energy of excitation, and neglect of neighbor anisotropy, the shift for the 13C car-bony1 carbon atom can be related directly to the local charge density at that carbon atom (14). Both theoretical and experimental results indicate that the chemical shift is directly proportional to the polarity of the carbonyl K bond.
A large downfield change in the chemical shift of ["Clcarbamyl-P is observed in the isomerized ternary complex of aspartate transcarbamylase with carbamyl-P and succinate, and also in the isomerized binary complex with [13C]PALA,' a transition state analog. Because of the established relationship between protonation of model compounds in hydrogen bonding systems and the direction of change in chemical shift, we can interpret our results with the enzyme in terms of protonation of the carbonyl groups in the active site. Alternative interpretations are also considered. MATERIALS  the shifts in all these solvents are upfield of the shift in water (12).
to [YZ]carbamyl-P, which was not purified from the reaction mixture. The equivalent weight was found to be 395 g/mol by complete enzymatic conversion to carbamyl-Laspartate, which was analyzed by the calorimetric method of Prescott and Jones (17) After re-evaporation, the residue was acidified to pH 1.5 for 2 h (to hydrolyze any phosphonamidates that may have formed) and, after the pH was readjusted to 8, the mixture in 250 ml was applied to a column of AG l-X8 as above. Peaks were located relative to marker ["CIPALA by analysis for phosphate (21) and the presumptive phosphonacetamide fraction was desalted on AG 50-X8. The melting point of the free acid form of phosphonacetamide agreed well with the value reported by Balsiger et al. (18). Analyses for C, H, and N were within 0.5% of theory for each element and the NMR spectrum showed only a single peak, with the expected splitting from coupling to 'IP. DIKEP and DIHOP-The preparation of DIKEP was described by Swyryd et al. (19). For reduction to DIHOP, three 6.mg portions of sodium borohydride were added at 20.min intervals to a 20 IUM solution of DIKEP (20 mg) at pH 8. The reduction was at least 92% complete, judging from the decrease in absorption at 275 nm (due to the carbonyl group). DIHOP was purified by chromatography on AG l-X8 and desalted on AG 50-X8 at 4' as described above. Purified DIHOP (2 pmol) was analyzed by co-chromatography with 0.4 pmol of radioactive PALA on a column of AG l-X8 (2 x 21 cm). DIHOP was eluted as a sharp peak one fraction earlier than PALA, indicating that it remains intact during purification.
Enzyme-Escherichia coli aspartate transcarbamylase and its catalytic subunit were prepared according to Gerhart and Holoubek (22)  shifts are reported as though they had been obtained at 25.2 MHz. Because of the long T, of the carbamyl-P carbonyl group (35 + 10 s) a small flip angle (10-15") was used for the radiofrequency pulse with a recycle time of 1.0 s. The free induction decay was multiplied by a negative exponential corresponding to 1 to 2 Hz line broadening prior to Fourier transformation. Difference spectra were calculated using Nicolet software.
["C]Urea (2 to 4 mM) was used as an internal standard.
The chemical shift of its carbonyl group was found to be invariant as a function of pH in phosphate buffer by comparison with external benzene or dioxane. Samples in D,O buffers were contained in 12-mm tubes. For experiments in H20, a lo-mm tube containing the sample was inserted into a 12.mm tube containing D,O for the lock signal. An aliquot of 200 mM carhamyl-P was added to the enzyme solution and the resulting mixture was kept cold until placed in the spectrometer.
Since the total time required for an experiment with carbamyl-P was 2 to 12 h, decomposition of carbamyl-P at the normal probe temperature of 33" would have been a major problem. The problem was circumvented by lowering the probe temperature to 23" and by using 40 mM phosphate buffer. Rosenbusch and Griffin (25) have found the half-life of carbamyl-P to be 20 h under these conditions. Spectra for [S'P]carbamyl-P were obtained at 40.5 MHz using a Varian XL-100 spectrometer.
The sweep width was 1000 Hz, the flip angle was 30", and the acquisition time 0.6 s. Inorganic phosphate of the buffer (40 mM) was used as an internal standard.
Since phosphate is known to have a pH-dependent chemical shift and since it binds to the enzyme, external HQO, was used to he sure that the phosphate shift was invariant at pH 7.0 with 1.0 mM enzyme, with and without the substrate.
Calculation of Bound Shifts-The quantitative criterion for fast exchange is that l/r, the reciprocal of the lifetime in the bound state, must be large compared to the chemical shift between the two states: l/r >> OKAY. For protonation of amide and urethan carbonyls the chemical shifts are about 3.4 ppm (86 Hz) (see below). Hence, for fast exchange it is necessary that l/r > > 540 ss'. Temperature-jump studies give an estimate of the second order rate constant for binding of carbamyl-P of 2.4 x lo* Mm'. s-' (29). Assuming that this constant is the same for the analogs, the known dissociation equilibrium constants can be used to compute the rate constant for dissociation of each compound: 1.7 x lOa s-' for carbamyl-P, 1.6 x lo5 so' for phosphonacetamide, and 14.4 s-l for PALA. Therefore, carbamyl-P and phosphonacetamide will be in fast exchange and PALA will be in slow exchange, i.e. separate peaks should be observed for the bound and free species. Free and bound PALA would still be in slow exchange even if the chemical shift were as small as 10 Hz. In the fast exchange case, the observed chemical shift is given by where JEC and 6, are the chemical shifts of bound and free carbamyl-P respectively, and (EC,)I(C,) and (C)/(C,) are the fractions of those species. Shifts from the free carbamyl-P peak are expressed as where AEC = (dEC -6,) is defined as the chemical shift for bound As an example, with 2 mM carbamyl-P, 1 mM active sites, 40 mM potassium phosphate, 10 mM succinate in D,O at apparent pH 7.0, with K,, (carbamyl-P) = 7 PM (31), K, (phosphate) = 1 mM (31), K,, (succinate) = 1.7 mM and K, (succinate) = 0.25 mM, 97% of the enzyme is in the ternary complex, 2% is in the binary complex with carbamyl-P, 1% is in the binary complex with phosphate, and less than 0.1% is in the binary complex with succinate.
For a dicarboxylic acid such as L-malate, where the interaction of the acid with free catalytic subunit (K,, = 1.53 mM) is stronger than the interaction with the subunit.carbamyl-P complex (K, = 10.4 mM), the fraction of subunit.r.-malate rises to 8% under our experimental conditions.

RESULTS
Dissociation Constants for Dicarboxylic Acids-Hey& et al.
(32) and Jacobson and Stark (27) have shown that dicarboxylic acids bind to free catalytic subunit in competition with carbamyl-P in Hz0 at pH 8.0. In order to calculate concentrations of the various complexes in NMR experiments, we determined constants for dissociation of dicarboxylic acids from catalytic subunit alone and from the subunit.carbamyl-P complex in ILO at apparent pH 7.0 ( Table I). The constant for dissociation of succinate from the ternary complex with carbamyl-P (K,) is substant,ially smaller than the constant for dissociation of succinate from the binary complex (K,,). For D-malate, these two constants are nearly equal. All other dicarboxylic acids tested bind more tightly to the free subunit than to the complex with carbamyl-P. if-urea 0 Hz [13C]Carbamyl-P and Catalytic Subunit-The chemical shift for free carbamyl-P in phosphate buffer in DzO at apparent pH 7.0 is 156.3 + 0.5 Hz upfield of internal urea or 160.8 ppm downfield of external tetramethylsilane ( Fig. la). This signal is a doublet with carbon coupled to "P by 4.5 Hz. Carbamyl-P is fully ionized above pH 7 and the chemical shift does not vary between pH 7.0 and 9.5. When catalytic subunit is added, there is a small but reproducible upfield change in the shift of 0.5 to 1.0 Hz (Fig. 1 b), corresponding to a change of 2 Hz (0.1 ppm) for the bound species. This small upfield change indicates that the environment of the carbonyl group of carbamyl-P in the active site is slightly different than in water.
[Y]Carbamyl-P, Catalytic Subunit and Succinate-When succinate is added, a downfield change in the shift is observed for carbamyl-P (Fig. lc). The magnitude of the change depends on the concentration of succinate (Table II). The large change for carbamyl-P bound in the ternary complex at apparent pH 7.0, 15 to 16 Hz, may indicate that the binding of succinate causes the carbonyl group of carbamyl-P to interact with a proton donor of the enzyme, as discussed below. The change in shift for carbamyl-P in the ternary complex decreases with increasing pH, suggesting that the complex is being titrated (Table III)." Beard and Schmidt (4) have observed a pH-' The pK. values of weak acids are 0.5 to 0.7 unit higher in D1O (33) and the pK, of imidazole is 0.4 unit higher (34). The effect of D,O on Carbamyl-P 156 Hz dependent relaxation of succinate protons upon the binding of succinate to the catalytic subunit carbamyl-P complex and suggest that two protonated groups which affect succinate binding are titrated between pH 7 and pH 10 and that there is an isomerization of a ternary complex. As shown in Fig. 2, there are two binary complexes of catalytic subunit and carbamyl-P in fast exchange with succinate and a conformational isomer of the ternary complex EHJ in which the ligands are tightly bound, (EHJ)'. Beard and Schmidt propose that a group on the enzyme with pK 8.2 is directly involved in the binding of succinate, and that a group with pK 6.9 is involved in the conformational change. We have fit our 13C data to this  model by assuming that the change in shift for bound carbamyl-P in EHI and EHJ is -2 Hz, the value for the change in the binary complexes, i.e. the isomerized complex (EHJ)' is the only species in which bound carbamyl-P experiences a large downfield change in chemical shift. The fraction of ternary complex isomerized can be evaluated as a function of pH at 23" from the distribution equations and optimized constants of Beard and Schmidt (4). From the relative amount of (EHJ)' at apparent pH 7.0, the change in shift for carbamyl-P in the isomerized complex is calculated to be 17.7 * 1.0 Hz (Table III). This value is not significantly different up to apparent pH 8.3, i.e. the observed change stays proportional to the amount of (EHJ)'. The fit is good at pH 7.3 and 7.6; at Subunit-The chemical shift for the amide carbonyl group of free PALA is extremely pHdependent because it is sensitive to ionization of the phosphonate group (Fig. 3). The second phosphonate proton dissociates with pK, about 7.5; the signal for PALA trianion is 187.8 Hz and that for PALA tetra-anion 235.8 Hz downfield of internal urea. At pH 7.0, the chemical shift for PALA is about 200 Hz, indicating that about 25% of free PALA is in the tetra-anion form at this pH. Like the carbamyl-P signal, the signal for PALA is a doublet. The coupling with 3'P is slightly pHdependent (4 Hz at pH 7.0, 3.5 Hz at pH 8.4).
Since PALA binds very tightly to the enzyme (K, = 2.7 x lOma M at pH 7 and 28" (20)), free and bound PALA should be in slow exchange. At pH 7.0, with about 2 eq of PALA per active site, two peaks with roughly comparable linewidths and intensities are observed, consistent with slow exchange (Fig. 4A). The upfield peak is at the position of free PALA at pH 7.0 and it increases in intensity with increasing concentration of free PALA. The other peak, 20 Hz downfield, is due to bound PALA. Since the peak for bound PALA is partially obscured by the envelope of signals from the enzyme carbonyl groups (   The solid line is a theoretical titration curve for pK = 7.5 and a total shift of 48 Hz. 4B), the contribution of the enzyme was subtracted to yield a difference spectrum (Fig. 4C).
['3C]Phosphonacetamide and Catalytic Subunit-The car-bony1 group of free phosphonacetamide has a chemical shift which is also extremely pH-dependent due to phosphonate ionization. The pK, for dissociation of the second phosphonate proton is 7.1 and the chemical shift of the dianion is 85 Hz downfield of the shift for the monoanion.
At pH 6.94, the coupling constant with 31P is 4.7 Hz. Phosphonacetamide competes with carbamyl-P, with K, = 0.66 mM at pH 8.0 (31); bound and free phosphonacetamide are in fast exchange when enzyme is present (40). A single resonance was observed, consistent with fast exchange, in two experiments with phosphonacetamide (4.3 and 1.5 mM) in the presence of catalytic subunit (0.8 mM) at apparent pH 6.94 in DzO, where 41% of free phosphonacetamide is in the dianion form. The observed change in shift was small, and the change for bound phosphonacetamide was calculated to be 5 to 7 Hz upfield of that for free phosphonacetamide at the same pH ( field of internal urea). Because the observed shift is very sensitive to small variations in pH in this case, the pH of the enzyme solution was checked carefully before and after each experiment and found to be constant within 0.01 unit. Interaction with DIKEP and DIHOP-The contribution of the amide carbonyl group of PALA to affinity for catalytic subunit was investigated with the analogs DIKEP, in which the carbonyl group is that of a ketone, and DIHOP, in which the ketone has been reduced to an alcohol. The K, values at pH 8 are 6.5 x lo-' M for DIKEP and 4 x lo-" M for DIHOP, each value based on the total concentration of all the optical isomers. The K, for DIHOP may be even higher than 4 x 10m5 M, since contamination by 1 to 2% of DIKEP, which would account for all the inhibition observed, cannot be ruled out. Catalytic subunit was titrated with DL-DIKEP by ultraviolet difference spectroscopy according to the procedure of Collins and Stark (20) in 40 mM glycylglycine buffer at pH 7.0. The magnitude of the difference spectrum at saturation was the by guest on March 23, 2020 http://www.jbc.org/ Downloaded from lsC NMR and Aspartate Transcarbamylase same as the magnitudes of those obtained with PALA or position of the 'YZ atom, thus causing upfield or downfield carbamyl-P plus succinate, with a major peak at 288.8 nm and changes in the chemical shift. Spectroscopic data (1,43,44) a minor peak at 281.1 nm. If only L-DIKEP binds tightly, indicate that there are likely to be aromatic rings in the active titration yields a value of 3.3 binding sites per catalytic trimer, site, and Schmidt et al. (40) proposed that the downfield in excellent agreement with the 3.1 binding sites found in a change in chemical shift observed for the protons of methyl parallel titration with PALA. Therefore, the Kr for L-DIKEP is phosphonate upon binding to the catalytic subunit, much 3.2 x lo-' M, about 5 times higher than the K, for PALA (6 x larger than the change for the protons of acetyl phosphate, lo-' M under the same conditions (27)). A similar difference might be due to the proximity of an aromatic ring edge-on to between PALA and DIKEP has been found with hamster as-the methyl group. If the edge of such a ring were to approach partate transcarbamylase (19).
the carbonyl group of carbamyl-P as a result of the conforma-Changing the carbonyl group of DIKEP to the hydroxyl of tional change induced by the binding of succinate, it might DIHOP has a much greater effect on binding than changing the well cause the effect we observe. It is difficult to choose amide -NH-of PALA to the -CH,-of DIKEP. Either definitively between ring current and protonation on the basis the carbonyl groups of both PALA and DIKEP make similar of the current NMR evidence alone, but an aromatic ring near important positive contributions to binding or the hydroxyl the carbonyl group of carbamyl-P would not aid catalysis, group of DIHOP makes an important negative contribution.
whereas protonation of the carbonyl oxygen atom is a most Since the carbonyl oxygen of a ketone is less basic than that of attractive mechanistic possibility; therefore we favor it. With an amide by a factor of about lo6 (41), it is most unlikely that L-DIKEP, protonation is much less likely because of the lower there is appreciable protonation of the bound DIKEP carbonyl pK, of the ketone carbonyl, and yet the conformational change group at pH 7, although a hydrogen bond to the proton donor with the catalytic subunit is indistinguishable from the one is not unlikely. We conclude that the hydroxyl group of DIHOP obtained with PALA. Therefore, 13C NMR experiments with encounters significant steric hindrance in the isomerized com-L-DIKEP should be very helpful in choosing between protonaplex, or that tetrahedral geometry at the site of the carbonyl tion of the carbonyl group and other mechanisms. group interferes with binding, or both.
In the catalytic mechanism we have proposed previously (1, 3) a substantial change in conformation occurs when dicar-DISCUSSION boxylic acids are bound, and substituents in the position of the Carbamyl-P-The carbamyl-P site of aspartate transcar-amino group of L-aspartate interfere both with affinity for the bamylase is known to be very accessible to a variety of ligands, enzyme and with the extent of conformational change upon since all compounds with a phosphonate or phosphate group saturation. Support for a large conformational change upon the tested inhibit the catalytic subunit competitively with respect binding of succinate has been obtained by difference spectrosto carbamyl-P (31). The small upfield change in the chemical copy (l), change in sedimentation coefficient (45), changes in shift of [Ylcarbamyl-P upon binding to the catalytic subunit circular dichroism (43) and optical rotatory dispersion (44), indicates that the environments of the carbonyl group in the temperature jump measurements (29), and proton NMR (4). binary complex and in water are quite similar. A previous The present results with ['%]carbamyl-P indicate that this study of the binding of phosphonacetamide to the catalytic conformational change may be accompanied by significant subunit by proton NMR indicated some sort of an interaction protonation of the carbonyl group. The variation of the between the enzyme and the carbonyl group of the inhibitor downfield change in chemical shift with pH fits very well a (40). In the case of carbamyl-P, a hydrogen bond to the enzyme model in which the isomerized complex (EH,I)' is the only similar to the hydrogen bond between water and the carbonyl species in which this protonation occurs. group of free carbamyl-P would be compatible with the 13C When other dicarboxylic acids are substituted for succinate, data.
smaller downfield shifts are observed (Table IV). By assuming Adding succinate to the binary complex causes a large that the downfield shift for [Yjcarbamyl-P occurs only in the downfield change in the chemical shift, which may result from isomerized complex (EH,I)' and that the magnitude of this partial protonation of the carbonyl group by the enzyme, shift is independent of the nature of the dicarboxylic acid, we perhaps by the hydrogen bond donor of the binary complex.
can calculate the fraction of ternary complex isomerized in Distortion of the amide bond, with resulting changes in the each case (Table V). For L-malate, the fraction isomerized is in hybridization of the carbon atom from sp' toward sp3, might reasonably good agreement with estimates from temperaturealso yield the downfield shift observed. Other possible causes jump and sedimentation experiments, each of which have been for the effect are changes in polarity, hydrogen bonds, and ring' carried out under somewhat different conditions (Table V). For currents. Since the chemical shift of urethan in aprotic solvents L-malate, the degree of conformational change estimated from less polar than water is upfield of the shift in water,' a decrease difference spectroscopy is much lower than estimates obtained in polarity in the active site is most unlikely to be responsible by the other methods and, for D-malate, difference spectra for the effects observed with carbamyl-P and PALA. A indicate extensive conformational change but the 13C NMR hydrogen bond from an enzyme donor in the ternary complex experiment shows little effect on bound carbamyl-P. Perhaps would have to increase the effect already present from hydro-the perturbation of tryptophan accompanying binding of a gen bonds between free carbamyl-P and water molecules to dicarboxylic acid to the binary complex reflects not only produce a change. Since only small changes are anticipated for conformational change but also changes in the polarity of the such a process on the basis of studies with model compounds environment of this chromophore caused by nearby ion pairs. (42), we consider it unlikely on quantitative grounds that the This possibility has been suggested previously by Griffin et al. effect we observe stems from this cause. Although the theoreti-(45) on the basis of circular dichroism data. Another possible cal basis for quantitating the magnitudes of ring current effects explanation is that the hydroxyl group of n-malate is near is still uncertain for 13C (42), the local magnetic field generated enough to the carbonyl group of carbamyl-P to provide some by the x electrons of aromatic rings may be parallel or shielding. anti-parallel to the applied field, depending on the relative Jacobson and Stark (27) have shown recently that both  Gerhart and Pardee (46). From the data of Table V it is apparent that these two analogs of L-aspartate also cause appreciable fractions of the ternary complex with catalytic subunit to become isomerized.
PALA and Phosphonacetamide-Interpretation of the 20-Hz downfield shift observed upon the binding of PALA to catalytic subunit is complicated by the necessity of doing the experiment at a pH near 7.0, since the pK, for dissociation of the second phosphonate proton is 7.5 and ionization is accompanied by a downfield change in chemical shift of 48 Hz. If the pK, for the phosphonate portion of PALA were to change to a value below about 6 upon binding, so that the only bound form of PALA at pH 7 were the phosphonate dianion, the chemical shift for PALA would be expected to move about 36 Hz downfield upon binding from this effect alone. Since a downfield change of 20 Hz is actually observed, a compensating change of 16 Hz upfield from some interaction at the carbonyl group of PALA is required, a result greatly different from the 17.5-Hz downfield shift observed for carbamyl-P in the presence of succinate. Alternatively, if the pK, for the phosphonate of bound PALA were changed to a value above about 8.5, the chemical shift for PALA would move 12 Hz upfield upon binding from this cause alone, and the observed change of 20 Hz downfield would imply a change of 32 Hz downfield from the interaction of the carbonyl group.
In order to distinguish among these two extreme possibilities and the intermediate cases in which the pK, for the phosphonate of bound PALA lies between 6 and 8.5, we examined the binding of phosphonacetamide to the enzyme, since only small effects would be expected from any interaction with the car-bony1 group of this compound, by analogy with the case of carbamyl-P in the absence of succinate. At apparent pH 6.94, the chemical shift for phosphonacetamide (pKa 7.1 in D,O) moves only about 6 Hz upfield upon binding, a small effect comparable to the ~-HZ upfield change seen with carbamyl-P (which is completely ionized at pH 7, so that there is no possible complication from a change in ionization state). The results with phosphonacetamide indicate strongly that the pK, of this compound is affected very little upon binding to the enzyme, i.e. the enzyme has about the same affinity for the mono-and dianion forms. If the enzyme also has approximately equal affinities for the trianion and tetra-anion forms of PALA, the downfield change of 20 Hz observed upon binding would reflect primarily a difference in the environment of the carbonyl group, similar to the change observed with carbamyl-P plus succinate. Of course, the conformations of the enzyme with PALA or phosphonacetamide bound are quite different. However, the 31P NMR data with carbamyl-P alone and in the presence of succinate indicate that the environment of this phosphate dianion changes much more when the binary complex is formed than it does when succinate adds to form the isomerized ternary complex.

Relative
Basicities of Carbonyl Groups and Changes in Chemical Shifts upon Proton&ion-Chemical shifts relative to urea are given in Table VI for carbamyl-P, urethan, PALA, and phosphonacetamide.
In order to interpret data for interactions of carbamyl-P and PALA with the catalytic subunit in terms of protonation of the carbonyl groups, we need to know something about their relative basicities and about how much their chemical shifts change upon full protonation. Both carbamyl-P and PALA are too labile in strong acid to determine these quantities directly, so values have to be inferred from the properties of model compounds such as urethan, butyramide, and phosphonacetamide, as described in Table VII. The downfield change in chemical shift of 17.7 Hz for carbamyl-P in the isomerized ternary complex (EHJ) represents 20% of the change obtained with urethan and phosphonacetamide upon full protonation. If the pK, of the phosphonate group of PALA is unaltered upon binding to the enzyme, the 20-Hz downfield change in chemical shift observed upon binding of PALA to catalytic subunit corresponds to about the same change with carbamyl-P and succinate. Since the pK, values for the carbonyl groups of PALA and carbamyl-P are about the same  where H, is the acidity function for amides in HCl (47). dThese values differ appreciably from the ones determined by Armstrong and Moodie (48) for urethan (pK, = -3.0) and butyramide (pK, = -1. o Observed change downfield from the species with fully protonated phosphonate groups.
'Too unstable at this concentration of acid to measure. g Calculated from the chemical shift of PALA in 38% DCl, assuming that the chemical shift for full protonation is the same as for the carbonyl group of phosphonacetamide.
n The pK. for carbamyl-P can be estimated in two ways. (a) Add the effect of substituting an oxygen for an amide methylene ( -0.8 pK unit) to the pK, for phosphonacetamide; (b) add the effect of the phosphonate group in going from butyramide to phosphonacetamide (~ 1.7 pK units) to the pK, of urethan. (Table VII) it seems quite reasonable for these groups to experience about the same change in the isomerized complexes.
We would like to know pK, values for the amide carbonyl groups of carbamyl-P and PALA when they are bound to the enzyme. Fully protonated enzyme-bound carbamyl-P would be NH,C+(OH)OP03=, and Table VII gives an estimate of the pK, for the species NH, C+(OH)OP03H2.
Of course, the first species can exist in appreciable concentration only in the active site of the enzyme, where specific acid catalysis is possible. Nevertheless, the inductive effects of -OPOs2-and -OPOsH, on the pK, of the carbonyl group are bound to be quite different and should be taken into account, even if only approximately.
From the comparison of butyramide with phosphonacetamide in Table VII it can be seen that substitution of -CH,POIHZ for -CH,CH,CH, decreases the pK, by 1.7 units. Comparable inductive effects are seen for substitutions of -CH,CO,H for -CH,CH,CH, (48). As a rough guide, the pK, of the NH,+ group in a peptide +NH,CH(R)C(O)NHR' is about 2 units lower than in the corresponding amino acid +NH,CH(R)CO,-.
Therefore, we estimate that the pK, for the species NH, C+(OH)OPO,= is about -4 or -3 and that the change in pK, required to achieve protonation 20% of the time at pH 7 is 9 or 10 units. An alternative model is that partial protonation occurs all the time, with an effect on the chemical shift 20% of that for full protonation. The energy to drive such a process need not be 20% of the energy to drive full protonation, so the change in pK, estimated above can be regarded as an upper limit.
Conclusions-A modified stepwise scenario can be proposed for the action of aspartate transcarbamylase by combining the present results with previous evidence summarized with references by Jacobson and Stark (3). The binding of the substrates is ordered. Carbamyl-P, the first substrate to bind, interacts first with a readily accessible site on the enzyme through its phosphate group, followed by a conformational change in which a hydrogen bond is formed between a proton donor of the enzyme and the carbonyl group. Close proximity of the enzyme to the NH2 group helps to hold the bound carbamyl-P rigidly. L-Aspartate then binds, causing a second conformational change in which the NH, group is pushed toward the carbonyl group of carbamyl-P. The aspartate is held rigidly by both carboxylates and also by close steric interactions with the enzyme, so that compression of the two substrates takes place only in a productive direction, along the reaction coordinate. During the conformational change, the hydrogen bond to the carbonyl group of carbamyl-P becomes shorter. Protonation or perhaps deformation of the amide activates this carbonyl group, which is otherwise very unreactive toward nucleophiles. This activated form of carbamyl-P must be well protected from solvent in isomerized complexes, since the catalytic subunit does not stimulate the decomposition of carbamyl-P in the presence of succinate.6 The pK, values of the carbonyl groups of PALA and carbamyl-P are similar, so that each may be protonated to about the same extent on the enzyme. In the case of DIKEP, shortening of a hydrogen bond to the carbonyl groups should also take place upon compression, but to the extent that protonation is important we would expect the effect on the ['"Cl chemical shift of this ketone to be much less, since a ketone is a much weaker base than the amides of PALA or carbamyl-P. Experiments with '%-enriched acetyl-P in the presence of succinate might help to reveal whether distortion of the amide bond of carbamyl-P plays a role in activating the carbonyl group. Fourier transform infrared measurements with the "C-labeled compounds we have used would also be quite informative.
Energy to drive both the conformational change of the enzyme and the proposed highly unfavorable shortening of the hydrogen bond (protonation of the carbonyl group) must of course be derived from unrealized potential binding energy of the substrates. Although the estimate is very rough, the pK, values of the carbonyl groups of carbamyl-P and PALA may be increased by as much as 9 or 10 units in the isomerized complexes. Since the observed affinity of the enzyme for PALA is about 10' Mm' at pH 7, PALA could bind with an affinity of 1Ol7 M-* or so in the absence of conformational change and carbonyl interaction, corresponding to a binding energy of about -23 kcal/mol at 25". Although this energy seems very large, it is comparable to recent estimates of some actual binding energies (the binding of avidin to biotin has a free energy of -20 kcal/mol) and to calculations of potential binding energies in the absence of conformational change (see Jencks (50) for a recent discussion in depth). In the case of PALA, an increase in the rotational and translational entropy of the system probably makes an important contribution.
For example, if three acetate anions were displaced from the active site upon the binding of one PALA, there would be a net gain of entropy for 2 small molecules. To a first approximation, this BM. 0. Modebe and G. R. Stark, unpublished results. could contribute 40 entropy units, or about ~ 12 kcal/mol to the free energy of binding. The nature of the postulated proton donor is unknown, but it should be noted that Beard and Schmidt (4) provide evidence that a group with pK, 7 is involved in isomerization of the ternary complex, and Greenwell et al. (51) have implicated the presence of 2 histidine residues in the active site through a study of the photooxidation of subunit. pyridoxal-P complexes. Decisive information on the nature of the catalytic residues and on the possible role of ring currents in the effects we have observed with Y-labeled compounds should come eventually from crystallographic maps of aspartate transcarbamylase (52), perhaps in the presence of PALA.