On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.     

Zn(II) coordination domain mutants of T4 gene 32 protein.

Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II)-substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less than or equal to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P. In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B). Spin-echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His-g32P-(A+B) [Pan, T., Giedroc, D.P., & Coleman, J.E. (1989) Biochemistry 28, 8828-8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS)     

Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis.

Site-specific mutagenesis has been used to probe amino acid residues proposed to be critical in catalysis by Escherichia coli asparaginase II. Thr12 is conserved in all known asparaginases. The catalytic constant of a T12A mutant towards L-aspartic acid beta-hydroxamate was reduced to 0.04% of wild type activity, while its Km and stability against urea denaturation were unchanged. The mutant enzyme T12S exhibited almost normal activity but altered substrate specificity. Replacement of Thr119 with Ala led to a 90% decrease of activity without markedly affecting substrate binding. The mutant enzyme S122A showed normal catalytic function but impaired stability in urea solutions. These data indicate that the hydroxyl group of Thr12 is directly involved in catalysis, probably by favorably interacting with a transition state or intermediate. By contrast, Thr119 and Ser122, both putative target sites of the inactivator DONV, are functionally less important.     

Impact of alterations near the [NiFe] active site on the function of the H(2) sensor from Ralstonia eutropha

In proteobacteria capable of H(2) oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H(2). The H(2)-sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni-Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H(2)-oxidizing and D(2)/H(+) exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para-/ortho-H(2) conversion activity showed that these centres were still able to bind H(2). This indicates that H(2) binding at the active site is not sufficient for the regulatory function of H(2) sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy-linked [NiFe]-hydrogenases. The respective RH mutant protein showed only traces of H(2)-oxidizing activity, whereas its D(2)/H(+)-exchange activity and H(2)-sensing function were almost unaffected. H(2)-dependent signal transduction in this mutant was less sensitive to oxygen than in the wild-type strain. These results suggest that H(2) turnover is not crucial for H(2) sensing. It may even be detrimental for the function of the H(2) sensor under high O(2) concentrations.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.     

Investigation of the contribution of histidine 119 to the conduction of protons through human Nox2.

The conduction of protons through human Nox2 has previously been shown to be dependent upon His115. Alignment of sequences for both animal and plant Nox proteins indicated that histidines 115 and 119 are both highly conserved, while His111 was conserved among animal homologues of Nox1-4. To investigate the possible role that these histidine residues might play in the conduction of protons through Nox2, we have introduced both paired and single mutations into these histidine residues. Each construct was used to generate a CHO cell line in which the expression of the mutated Nox2 was assessed. Nox2 was expressed in each of the CHO cell lines generated, however, the level of expression of H111/115L in CHO cells was lower and that of H111L very much reduced, compared to that of wild-type Nox2. The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2. Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L. In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone. Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression. We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2. His119 has been identified as a highly conserved residue for which no function has previously been proposed.     

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.     

Proton, calcium, and magnesium binding by peptides containing gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid (Gla) is believed to bind Ca [II] ions and Mg [II] ions in prothrombin and other coagulation proteins. Binding constants for H+, Ca [II] ions, and Mg [II] ions to Gla-containing peptides are determined using pH and ion selective electrode titrations. The binding constants for peptides containing a single Gla residue are similar to the constants for malonic acid. Peptides containing two Gla residues in sequence (di-Gla peptides) bind Ca [II] ions and Mg [II] ions more strongly. KMgL for the di-Gla peptides is similar to the site-binding constant for Ca [II] ions in denatured BF1. These di-Gla peptides may be useful analogs for metal binding by the disordered Gla domain in BF1.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.     

The roles of ATP4- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme. From the change in the chemical shifts of the 'basic-patch' histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1'H resonances it is apparent that there are at least two ATP-binding sites on the enzyme. Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic-patch region of the N-terminal domain. The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein. Evidence from previous two-dimensional NMR experiments [Fairbrother et al. (1990) Eur. J. Biochem. 190, 161-169] suggests that the secondary site is equivalent to the crystallographically observed catalytic site. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration. The possible importance of these observations in the regulation of this enzyme in vivo are discussed.     

Histidine pK(a) values for the N-lobe of human transferrin: effect of substitution of binding site Asp by Ser (D63S)

The pK(a) values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe(3+)-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pK(a) values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the varepsilonCH resonance of His249. For the wild-type protein, the His residue with a pK(a) of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pK(a) value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pK(a) value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from log K 4.0 to log K 3.3. (1)H NMR spectral changes induced by Ga(3+) binding to the mutant are compared to those observed for the wild-type protein.     

Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

His ... Asp catalytic dyad of ribonuclease A: histidine pKa values in the wild-type, D121N, and D121A enzymes

Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.     

Novel Structure–Function Information on Biogenic Amine Transporters Revealed by Site-Directed Mutagenesis and Alkylation

The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [³H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements.

Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N epsilon, N epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-2H]cytochrome c or more selective labeling of just histidine residues in [epsilon-2H]histidine cytochrome c. Deuterium NMR measurements on [N epsilon, N epsilon-C2H3]lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The [amide-2H]cytochrome c provided "immobile" amide spectra showing that only the most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of [amide-2H]cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on [epsilon-2H]histidine cytochrome c in solution showed that all labeled histidine residues were also "immobile" compared with the overall reorientational motion of the protein. The interaction with cardiolipin bilayers appeared to create a high degree of mobility for the side-chain sites of [N epsilon, N epsilon-C2H3]lysyl cytochrome c and perturbed backbone structure to instantaneously release all deuterons in [amide-2H]cytochrome c. The [epsilon-2H]histidine cytochrome c derivative, when complexed with cardiolipin, failed to produce any detectable wide-line 2H NMR spectrum, demonstrating that the overall reorientational motion of bound protein was not isotropic on the NMR time scale, i.e., tau c greater than 10(-7)s.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.     

Assignment of tyrosine resonances in the 1H-NMR spectrum of tryptophan synthase alpha-subunit. Monitoring conformational changes due to substitutions at position 49

In order to monitor the conformational changes of tryptophan synthase alpha-subunit from Escherichia coli in solution resulting from amino acid substitutions, we have assigned the Tyr resonances in the aromatic region of the 1H-NMR spectrum to specific residues. In the spectrum of the alpha-subunit deuterated with [2,3,4,5,6-2H5]Phe and [3,5-2H2]Tyr, the C2 and C6 protons of Tyr gave completely isolated signals at acidic p2H. Some of the C3 and C5 proton resonances overlapped with each other at acidic p2H. By using a series of mutant alpha-subunits in which each Tyr was singly substituted with His or Phe, we can now assign each of seven Tyr resonances in the aromatic region to a specific residue. We have previously studied the conformational stability of a series of variant alpha-subunits at position 49 [Yutani et al. (1987) Proc. Natl Acad. Sci. USA 84, 4441-4444]. We now compare the 1H-NMR spectra in the aromatic region of the wild-type alpha-subunit and mutant alpha-subunits substituted with Phe or Gly in place of Glu-49. The results suggest that the major conformational effects of substitutions at position 49 are localized close to the position of substitution.