Effects of internal motions on the development of the two-dimensional transferred nuclear Overhauser effect

Summary In this paper we address the influence of internal motions on the development of the transferred nuclear Overhauser effect in a ligand undergoing chemical exchange between a free and a bound state. We examine the effects of varying the effective correlation time as well as the motional order parameter for methyl group and phenyl ring rotations in the free and bound ligand conformations. The effect of decreasing the motional order for a proton pair on a methyl group or phenyl ring is to decrease the effective correlation time of the internuclear vector, and thus to decrease the cross-relaxation rate between the proton pair. This functions to dampen the effects of spin diffusion, especially in the bound ligand- where cross-relaxation rates are much faster than in the free ligand. The effect of changing the effective correlation time for methyl group motions has little effect on the build-up behaviour of the transferred nuclear Overhauser effect for small values of fraction bound, but a larger effect on how fast it decays. This effect is greater for internal motions in the free peptide than it is for internal motions in the bound peptide.Dedicated to the memory of Professor V.F. Bystrov     

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.     

Binding modes of inhibitors to ribonuclease T1 as elucidated by the analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP or 5'GMP were assigned to specific amino acid residues by 2D-NMR spectra in comparison with the crystal structure of RNase T1-2'GMP complex. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take syn form as a bound conformation, while 5'GMP takes anti conformation around glycosidic bonds.     

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.     

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton–aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton–ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.     

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.     

A transferred NOE study of a tricyclic analog of acyclovir bound to thymidine kinase

Summary A purine derivative with an acyclic sugar analog, 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-6-ethyl-9-oxo-5H-imidazo[1,2-a]purine, was studied in the free state and in complex with herpes simplex virus thymidine kinase (HSV1 TK). Transferred NOE experiments, combined with a full relaxation matrix analysis of the substrate's spin system, resulted in a set of distance constraints for all proton pairs. These constraints were used in structure determination procedures based on simulated annealing and molecular dynamics simulations to obtain a family of structures compatible with the experimental NMR data. The results indicate that, although in both states the chains have the syn orientation with respect to the aromatic rings, in the free state the substrate's acyclic moiety is relatively disordered, while in the bound state only one specific conformation is preferred. Fluctuations can only be seen in the case of the terminal hydroxyl group, for which no NOE was recorded and hence no constraints were available.     

Relation of Raman order parameters to spin labeling parameters

For the quantitation of Raman and spin labeling data order parameters are commonly used. The spin label order parameter measured at any depth in the layer is a weighed sum of the segmental order since, due to fast conformational interconversions, each CH₂ segment is partly in trans and partly in non-trans, e.g. gauche, kink, jog, etc., conformation during the measurement. The weighing factor, the trans finding probability, varies along the chain (cf. flexibility profile) but its mean value should be equal to the Raman trans order parameter. This correlation is illustrated with the experimental data obtained for dipalmitoyl phosphatidylcholine and n-alcohol mixtures. The rate of rotational diffusion, a dynamical parameter from spin labeling studies, is correlated with the lateral packing density as measured by the Raman lateral order parameter. For the obtained linear correlation a qualitative explanation is given.The effect of a series of long chain alcohols on the phase transition characteristics of dipalmitoyl phosphatidylcholine was investigated. The possible role of hydrogen bonding in the interfacial region is emphasized.     

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 A resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4' carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.     

Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI)

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.     

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.     

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-nmr spectroscopy

¹³C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin–lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about, 0.1–0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do-occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups. © 1993 John Wiley & Sons, Inc.     

Conformational changes in cytochromeaa 3 and ATP synthetase of the mitochondrial membrane and their role in mitochondrial energy transduction

1. The thermodynamics and molecular basis of energy-linked conformational changes in the cytochrome aa3 and ATP synthetase complexes of the mitochondrial membrane have been studied with spectrophotometrical and fluorometrical techniques. 2. Ferric cytochrome aa3 exists in two conformations, high spin and low spin, the equilibrium between these states being controlled by the electrical potential difference across the mitochondrial membrane. The conformational change is brought about by an electrical field-driven binding of one proton per aa3 to the complex. At pH 7.2 the concentration of the two conformations is equal at a membrane potential of 170 mV corresponding to about 4 kcal/mole. 3. The high to low spin transition in ferric aa3 is also induced by hydrolysis of ATP in which case two molecules of aa3 are shifted per ATP molecule hydrolyzed. This is in accordance with translocation of two protons across the mitochondrial membrane coupled to hydrolysis of ATP as proposed in the chemiosmotic theory of oxidative phosphorylation. 4. The conformational transition in cytochrome aa3 is not an expression of the formation of a 'high-energy' intermediate or reversal of the energy-transducing pathway of oxidative phosphorylation, but is presumably the basis of allosteric control of the activity of cytochrome oxidase by the energy state of the mitochondrion. This control is exerted by a regulatory mechanism in which the electrical potential difference controls the conformation and redox properties of the heme centres and thereby the rate of oxygen consumption. 5. The synthesis of one molecule of ATP by oxidative phosphorylation is energetically equivalent to the work done in carrying two electrical charges across the entire mitochondrial membrane. 6. Fluorescence changes of aurovertin bound to ATP synthetase reveal that the electrical membrane potential induces a conformational change in the F1 portion of the enzyme which is probably associated with dissociation of the natural F1 inhibitor protein. This conformational change is energetically equivalent to the work done in carrying one electrical charge across the mitochondrial membrane. 7. A model is proposed for the mechanism of the electrical field-induced conformational changes in the cytochrome aa3 and ATP synthetase complexes, and the significance of these changes in the mechanism and control of mitochondrial energy conservation is discussed.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate.

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

1H-NMR study of heme propanoate mobility in the active site of myoglobin from Galeorhinus japonicus

Time-dependent NOE studies of the C13(1) and C17(1) methylene proton resonances of the heme peripheral propanoate groups have elucidated their mobility in the active site of the ferric high-spin form of Galeorhinus japonicus myoglobin. A large difference in the chemical shift due to the non-equivalence of the heme C13(1) and C17(1) methylene proton resonances allows selective irradiation of a given proton resonance by a high-power selective decoupler pulse in spite of their fast relaxation rates. NOE accumulation of the resonance of one methylene proton after saturation of the resonance of the other proton essentially follows the theoretical prediction derived using the two-spin approximation, and the cross-relaxation rates for the heme C13(1) and C17(1) methylene proton spin systems were quantitatively determined. The correlation time for the mobility of the internuclear vector connecting the heme C13(1) or C17(1) methylene protons was then calculated from the cross-relaxation rate and values of approximately 11 ns were obtained for both C13(1) and C17(1) methylene groups in 2 mM Galeorhinus japonicus myoglobin at 35 degrees C. The immobile C13(1) and C17(1) methylenes of the heme propanoate groups, together with a large difference in chemical shift between the methylene proton resonances, dictate their fixed orientation with respect to the protein moiety as well as the heme plane, and are therefore consistent with the immobile heme in the active site of myoglobin.     

Presence of endogenous calcium ion and its functional and structural regulation in horseradish peroxidase

The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study.

The replacement of Arg-54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000-fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592-2600). In the X-ray structure of the wild-type enzyme, Arg-54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85, 4205-4208). The Arg-54-->Ala enzyme was crystallized in the presence of the transition state analog N-phosphonacetyl-L-aspartate (PALA), data were collected to a resolution limit of 2.8 A, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P-NMR studies indicate that the binding of CP to the wild-type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa approximately 7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg-54-->Ala enzyme is significantly narrower than that of CP bound to the wild-type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered. 31P-NMR studies of PALA complexed to the wild-type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg-54-->Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg-54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.