Biochemical and structural studies of N5-carboxyaminoimidazole ribonucleotide mutase from the acidophilic bacterium Acetobacter aceti

N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (PurE) catalyzes the reversible interconversion of acid-labile compounds N5-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on its adaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichia coli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable at pH 3.5, with a > or = 20 degrees C higher thermal unfolding temperature at all pHs. His89 is not essential and does not function as part of a proton relay system. The kcat pH-rate profile was consistent with the assignment of pK1 to unproductive protonation of bound nucleotide and pK2 to deprotonation of His59. A 1.85 A resolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonation of CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme and a water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotide decarboxylation in the forward direction (N5-CAIR --> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., and Stubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in the reverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essential role of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO2-azaenolate intermediate.     

N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis

N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.     

Treponema denticola PurE Is a bacterial AIR carboxylase

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Heteronuclear 2D (1H-13C) MAS NMR Resolves the Electronic Structure of Coordinated Histidines in Light-Harvesting Complex II: Assessment of Charge Transfer and Electronic Delocalization Effect

In a recent MAS NMR study, two types of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila were resolved: Type 1 (neutral) and Type 2 (positively charged) (Alia et al. J. Am. Chem. Soc. ). The isotropic (13)C shifts of histidines coordinating to B850 BChl a are similar to fully positively charged histidine, while the (15)N shift anisotropy shows a predominantly neutral character. In addition the possibility that the ring currents are quenched by overlap in the superstructure of the complete ring of 18 B850 molecules in the LH2 complex could not be excluded. In the present work, by using two-dimensional heteronuclear ((1)H-(13)C) dipolar correlation spectroscopy with phase-modulated Lee-Goldburg homonuclear (1)H decoupling applied during the t(1) period, a clear and unambiguous assignment of the protons of histidine interacting with the magnesium of a BChl a molecule is obtained and a significant ring current effect from B850 on the coordinating histidine is resolved. Using the ring current shift on (1)H, we refine the (13)C chemical shift assignment of the coordinating histidine and clearly distinguish the electronic structure of coordinating histidines from that of fully positively charged histidine. The DFT calculations corroborate that the coordinating histidines carry approximately 0.2 electronic equivalent of positive charge in LH2. In addition, the data indicate that the ground state electronic structures of individual BChl a /His complexes is largely independent of supermolecular pi interactions in the assembly of 18 B850 ring in LH2.     

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.     

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.     

Evidence for the direct transfer of the carboxylate of N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) to generate 4-carboxy-5-aminoimidazole ribonucleotide catalyzed by Escherichia coli PurE, an N5-CAIR mutase

Formation of 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) in the purine pathway in most prokaryotes requires ATP, HCO3-, aminoimidazole ribonucleotide (AIR), and the gene products PurK and PurE. PurK catalyzes the conversion of AIR to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) in a reaction that requires both ATP and HCO3-. PurE catalyzes the unusual rearrangement of N5-CAIR to CAIR. To investigate the mechanism of this rearrangement, [4,7-13C]-N5-CAIR and [7-14C]-N5-CAIR were synthesized and separately incubated with PurE in the presence of ATP, aspartate, and 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetase (PurC). The SAICAR produced was isolated and analyzed by NMR spectroscopy or scintillation counting, respectively. The PurC trapping of CAIR as SAICAR was required because of the reversibility of the PurE reaction. Results from both experiments reveal that the carboxylate group of the carbamate of N5-CAIR is transferred directly to generate CAIR without equilibration with CO2/HCO3- in solution. The mechanistic implications of these results relative to the PurE-only (CO2- and AIR-requiring) AIR carboxylases are discussed.     

Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.     

Changes in interhelical hydrogen bonding upon rhodopsin activation

Hydrogen bonding interactions between transmembrane helices stabilize the visual pigment rhodopsin in an inactive conformation in the dark. The crystal structure of rhodopsin has previously revealed that Glu122 and Trp126 on transmembrane helix H3 form a complex hydrogen bonding network with Tyr206 and His211 on H5, while the indole nitrogen of Trp265 on H6 forms a water-mediated hydrogen bond with Asn302 on H7. Here, we use solid-state magic angle spinning NMR spectroscopy to probe the changes in hydrogen bonding upon rhodopsin activation. The NMR chemical shifts of 15N-labeled tryptophan are consistent with the indole nitrogens of Trp126 and Trp265 becoming more weakly hydrogen bonded between rhodopsin and metarhodopsin II. The NMR chemical shifts of 15N-labeled histidine show that His211 is neutral; the unprotonated imidazole nitrogen is not coordinated to zinc in rhodopsin and becomes more strongly hydrogen bonded in metarhodopsin II. Moreover, measurements of rhodopsin containing 13C-labeled histidine show that a strong hydrogen bond between the side-chain of Glu122 and the backbone carbonyl of His211 is disrupted in metarhodopsin II. The implications of these observations for the activation mechanism of rhodopsin are discussed.     

Effect of pH on the iso-1-cytochrome c denatured state: changing constraints due to heme ligation

The effect of pH on the denatured state (3 M guanidine hydrochloride) was evaluated with fluorescence spectroscopy for four variants of iso-1-cytochrome c, AcTM (no surface histidines), AcH26 (surface histidine at position 26), AcH54 (surface histidine at position 54), and AcH54I52 (stabilizing I52 mutation added to AcH54). Changes in the compactness and the heme ligation of the denatured state, as a function of pH, were monitored through changes in Trp 59-heme fluorescence quenching. With the AcTM and AcH26 variants, no change in the fluorescence intensity occurs from pH 4 to 10. However, for the AcH54 and AcH54I52 variants the fluorescence intensity drops significantly between pH 4 and 6, consistent with His 54 binding to the heme of cytochrome c. Between pH 8 and 10 fluorescence intensity increases again, indicating that the His 54 is displaced from the heme. The data are consistent with lysines 4 and 5 being the primary heme ligands at alkaline pH, under denaturing conditions. This conclusion was confirmed by site-directed mutagenesis. Thermodynamic analysis indicates that heme-ligand affinity in the denatured state is controlled primarily by sequence position (loop size) and that when histidines are present they inhibit lysine ligation until approximately pH 8.5-9.0 as compared to pH 7.5 with the AcTM variant. Thus, at physiological pH, histidine ligands provide the primary constraint on the denatured state of cytochrome c. The heme-Trp 59 distance in the denatured state of iso-1-cytochrome c, derived from analysis by F?rster energy transfer theory, is approximately 26 A at pH 4 and 10, much shorter than the random coil prediction of 56 A. Surprisingly, the heme-Trp 59 distance in the His 54 bound conformation only drops to approximately 21 A, consistent with an extended conformation for the short polypeptide segment separating heme and Trp 59.     

Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway

BACKGROUND: Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE. PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR. PurEs from higher eukaryotes are homologous to E. coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly. RESULTS: The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry. A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit. The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit. Co-crystallization experiments with CAIR confirm this to be the mononucleotide-binding site. The nucleotide is bound predominantly to one subunit, with conserved residues from a second subunit making up one wall of the cleft. CONCLUSIONS: The crystal structure of PurE reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of PurE with CAIR, reveals the location of the active site. The environment of the active site and the analysis of conserved residues between the two classes of PurEs suggests a model for the differences in their substrate specificities and the relationship between their mechanisms.     

Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin is a low-spin complex whose endogenous hexacoordination gives rise to optical and NMR characteristics reminiscent of cytochrome b(5) [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. In this work, the sequential assignments using (15)N-(13)C-labeled protein, (1)H nuclear Overhauser effects, and longitudinal relaxation data identified His70 as the proximal histidine and His46 as the sixth ligand to the iron ion. It was also found that one of two possible heme orientations within the protein matrix is highly preferred (>90%) and that this orientation is the same as in vertebrate myoglobins. The rate constant for the 180 degrees rotation of the heme within a protein cage to produce the favored isomer was 0.5 h(-1) at 25 degrees C, approximately 35 times faster than in sperm whale myoglobin. Variable temperature studies revealed an activation energy of 132 +/- 4 kJ mol(-1), similar to the value in metaquomyoglobin at the same pH. The rate constant for heme loss from the major isomer was estimated to be 0.01 h(-1) by optical spectroscopy, close to the value for myoglobin and decades slower than in the related Nostoc commune cyanoglobin. The slow heme loss was attributed in part to the additional coordination bond to His46, whereas the relatively fast rate of heme reorientation suggested that this bond was weaker than the proximal His70-Fe bond. The standard reduction potential of the hexacoordinated protein was measured with and without poly-L-lysine as a mediator and found to be approximately -150 mV vs SHE, indicating a stabilization of the ferric state compared to most hemoglobins and b(5) cytochromes.     

13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.     

Two-dimensional NMR studies of staphylococcal nuclease: evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin system assignments of the aromatic amino acids in the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3',5'-bisphosphate at pH 5.1 (H2O) or pH 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to greater than 95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to greater than 95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H[13C]SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE). The aromatic protons of the spin systems were identified from 1H[13C]SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1H alpha-13C alpha or 1H beta-13C beta direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101] and 1H delta-13C delta direct cross peaks in the aromatic region of the 1H[13C]SBC-NOE spectrum. The His121 1H delta 2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H[13C]SBC, 1H[13C]MBC, and 1H[15N]MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3',5'-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183-201]. The unusual chemical shift of His121 1H delta 2 is consistent with ring current calculations from either X-ray structure.     

Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.     

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.     

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.     

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.