Spectroscopic probes of Escherichia coli glutamine synthetase

Biochimica et Biophysica Acta, 371 (1974) 432-441 O Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 36869 SPECTROSCOPIC PROBES OF E S C H E R I C H I A THETASE

COLI G L U T A M I N E SYN-

RARE E A R T H IONS BY D I F F E R E N C E ABSORPTION*

FREDERICK C. WEDLER and VITO D'AURORA Cogswell Laboratory, Chemistry Department, Rensselaer Polytechnic Institute Troy, N.Y. 12181 (U.S.A.)

(Received May 20th, 1974)

SUMMARY 1. Nd(III) and other tripositive lanthanide ions stimulate the biosynthetic activity of adenylylated Escherichia coli glutamine synthetase (L-glutamate:ammonia ligase (ADP), EC 6.3.1.2) about 10--20 ~o as well as Mn(II) at pH 6.50. With other forms of the enzyme and other assays, Nd(III) inhibits activity. 2. Probe experiments with EPR and pulsed NMR, suggest that Nd(III) binds to the Exl-(Mn2+)~ enzyme at the n2 sites to produce the E u - ( M n 2+ )12-( i Nd 3+ )~z n2 complex. 3. Nd(III) bound to adenylated enzyme, compared to free ion, produces a difference absorption spectrum with relatively sharp peaks and troughs in the 300600-nm region. 4. This difference spectrum has been used to qualitatively evaluate the nature of the metal ion environment, and as a probe for studying perturbations by small molecules bound to the protein. The unique spectral changes induced by each substrate or modifier supports the conclusion that each possesses a unique binding site or mode of interaction with the protein.

INTRODUCTION Glutamine synthetase (L-glutamate:ammonia ligase (ADP), EC 6.3.1.2) plays an important role in nitrogen metabolism. The characterization, structure, and function of this enzyme in Escherichia coli have been investigated in depth, especially with regard to regulation [1, 2] and detailed physical characteristics [3]. The dodecameric native enzyme contains identical subunits of molecular weight about 50 000, to each of which may be covalently linked an AMP group via a phosphodiester bond with a tyrosine residue [4]. This adenylylation alters the pH optimum of activity and sensitivity to bound modifiers. Specific stimulation by Mn(lI) or Mg(II) also depends * Portions of this work have been presented in preliminary form at the 164th ACS National Meeting, New York, August, 1972, and at The 9th International Congress of Biochemistry, Stockholm, Sweden, July, 1973, both in abstract form, and in the Proceedings of the 10th Rare Earth Research Conference, Carefree, Arizona, April, 1973.

433 on the type of subunit, adenylylated or not, respectively. The present study was undertaken to attempt to introduce a spectroscopic probe molecule at or near the active site of this intriguing enzyme. That Nd(III) can replace Mg(II) or Ca(II) at specific metal sites in certain systems has been adequately demonstrated with bovine serum albumin [5], trypsinogen [6], a-amylase [7, 8], transferrin [9], lysozyme [10], concanavalin A [11, 12], pyruvate kinase [12], membranes of mitochondrial systems [13] and muscles [14, 15], and to nucleic acids [16] and Staphylococcus nuclease [17]. The three different classes of divalent metal ion sites on E. coli glutamine synthetase have been studied and reviewed recently by Ginsburg [3]. In brief, the n~ sites (one/subunit) bind metal tightest and tighten the protein into its active or activatable conformation; the n, sites (one/subunit) must be filled to stimulate catalytic activity, presumably by complexation with substrates and protein; and the n3 sites (approx. four/subunit)are non-essential but may aid general protein stability. Recent N M R studies of water proton relaxation rates by bound Mn(II) have elucidated further details of metalsubstrate site juxtaposition [18]. Correlations with this latter information have aided interpretation of the present data, as have recent model optical spectral studies with amino acid, carboxylate, and other types of ligands bound to lanthanides (refs 19-23 and Darnall, D. W., personal communication). METHODS Glutamine synthetase was prepared from E. coli strain W cells by the procedures described by Shapiro and Stadtman [1]. Both adenylylated (Ell) and unadenylylated (E2) enzyme forms were obtained from separate batches of cells, and were shown by disc gel electrophoresis to be > 99 7o homogeneous after purification. Assays for activity were carried out by the biosynthetic (P~ release) and reverse transferase (7-glutamylhydroxamate formation) reactions, according to published procedures [I]. Absorption spectra were obtained with a Cary 14 double-beam recording spectrophotometer, equipped with a 0-0.01, 0.1-0.2 absorbance unit slidewire, and using both 1-cm and 5-cm path length cells. For optical difference absorption spectra, the rare earth ions were introduced into the protein by the following procedure. First, it was found that apoprotein (metal free), produced by dialysis against 100 volumes of 10 -3 M EDTA, was precipitated extensively upon exposure to Nd a+ at any pH over the range of 5.5 to 7.5, and at any reasonable concentration of protein. This behavior, it was thought, may result from an "over-tightened" conformation of dodecamer upon Nd(III) binding to the nl sites [3, 24]. Thus, Nd(III) was introduced to protein in which the nx sites were stoichiometrically occupied by Mn z+. Nd(III) at 5.10 -3 M then was dialyzed overnight into a 1 mg/ml solution of this protein (usually Exl) at pH 6.0, then the pH was raised carefully to 6.5 and the solution concentrated to 5 mg/ml on a collodian bag (Sartorius) device. These procedures avoided precipitation problems with either protein or Nd(III), but introduce certain other questions, which are discussed below. In assay procedures with rare earth ions present the lanthanide ions were pipetted directly into assay solutions containing enzyme at micromolar levels with M 2+ in n~ sites. Relaxed apoenzyme was produced by dialysis against two changes of 100 volumes of 1 mM EDTA, pH 6.5, or by gel filtration, as

434 described by Hunt et al. [25]. Mn(II) concentration was then determined by atomic absorption spectroscopy, and was found to be < 0.05 Mn(II) per subunit as a result of these procedures. Free Mn(II) in solution with enzyme was determined at 25 °C in the presence and absence of added Nd(III), 5 mM, by EPR spectroscopic techniques, using a Varian E-9 instrument. The enhancement of water proton relaxation rate in the presence of Eax-(MnZ+)12 and then with Nd(III) added to this species, as above, was determined with an NMR-Specialties variable frequency pulsed N M R spectrometer at 25 °C at 24 MHz. These latter conditions had been determined by extensive earlier experimentation on temperature and frequency dependence of (1/Tap) values [18] to be optimal for this approach. RESULTS

(1) Enzyme activation by rare earth ions The lanthanide series of trivalent cations was examined for the ability of each ion at 5 mM to stimulate biosynthetic activity in adenylylated apo-glutamine synthetase. The results of these experiments are presented in Fig. 1. The stimulation of activity increases somewhat as ionic radius becomes closer to that of Mn(II), approx. 0.80 ,~, the natural activating cation, but is still less than 20 ~ of that activity obtained with 5 mM Mn(II). Because of the rather extensive model ligand studies carried out with Nd(III) [19], this ion was selected for further activation and spectrophotometric probe investigations.

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(2) Effects of Nd(llI) on activity The effects of titration of adenylylated and unadenylylated enzyme with Nd(III) on activity, with and without Mn(II) in the n t sites is shown in Fig. 2. Because of the multiplicity of metal sites and effects possible, these experiments were carried out in lieu of competitive inhibition or similar studies that would otherwise be possible for an enzyme with a single metal site [17].

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Fig. 2. Activity responses of various forms of glutamine synthetase to titration by Nd 3+ with fixed levels of subunits (SU), Mnz+, and ATP or ADP, these concentrations as indicated by arrows straight downward. In later experiments (Figs. 3-5) spectra were determined with 5 mM NdCIs. l, Elz with biosynthetic assay (A, O) pH 7.6, 5 mM ATP, (B, •) pH 6.5, with 5 mM ATP, and (C, A) pH 6.5, l mM ATP; titration was also carried out with Mn2÷ (e) pH 6.5, with 5 mM ATP, scale reduced 10 x. II, Ez with biosynthetic assay, pH 7.5. Titration by Nd(lll) with 50 mM MgCI2 (©), 1 mM MnC12 (A) and 0.1 mM MnC12 (O). III, E , with transferase assay, pH 6.5, with minimal Mnz+. (A, circles): complete, standard levels of substrates. (B, triangles): substrate levels reduced 2.5 x. (C, crosses): substrate levels reduced 5 x, with constant ADP plus 1 mM MnC12. Basically, the titration curves indicate that biosynthetic activity of the adenylylated enzyme is stimulated by N d ( I I I ) at p H 6.5 or 7.5, b u t that unadenylylated enzyme activity is not, except at very high N d ( I I I ) levels, > 10 mM. Transferase activity of adenylylated enzymc is strongly inhibited by N d ( I I I ) also. The shapes of

436 these titration curves relative to ATP or ADP levels suggest that Nd(III) is complexing with nucleotide in the active site region, presumably at n2 sites. Some competition for sites occupied by Mn(II) or Mg(II) may also occur, but generally the levels of substrates present do not alter metal-protein binding constants appreciably. One may compare ionic radii in several ways to predict whether one ion will best substitute for another. In this case, Nd(III) substitutes better for Mn(II) than for Mg(II), which suggests that a comparison on the basis of radius/unit charge may be more valid than comparing ionic radii alone. In other cases, e.g. for systems involving Ca(II) [5-17], the latter method of comparison is obviously better.

(3) Location of Nd(III) on the enzyme It was important to know whether the binding of Nd(III) to the E11-(Mn2+)~, ~ complex leads to displacement of Mn 2+ from the nl sites or to formation of a species with Nd(III) bound elsewhere in the protein, most likely to n2 sites. This was explored by two separate approaches. First, the amount of free Mn(II) in a solution of Elt-( Mn 2+)12, ~1 with [enzyme subunit] = [Mn 2÷] = 1.0"10 -4 M, pH 6.5, was explored by EPR spectroscopy. Under these conditions, 94.0 ~ of Mn(II) was bound to the enzyme, and upon addition of 5 mM Nd(III) the value became 96.6 ~ of total Mn(II) bound. This implies that Nd(III) binding does not displace bound Mn(I1) from the nl sites. Second, the effect of bound Nd(III) on Mn(II) in the nl sites was probed by pulsed N M R studies on the relaxivity of water protons induced by bound Mn z+. This value (1/Tip) was 33.4 for 0.2 mM Mn-subunit at 24 MHz, 25 °C, pH 6.5; it decreased only to 32.5, or less than 3 ~ , upon addition of 6.5 mM NdCI3. These two results, taken together argue that Mn(II) remains in the nl sites in a relatively unperturbed state as Nd(III) binds to the protein, most likely at the n2 sites.

(4) Spectrophotometric studies From the data of Fig. 2, it was decided to study adenylylated enzyme at pH 6.5 with 5 mM Nd(III) bound. At protein levels above 5 mg/ml, Nd(III) above 5 mM, or pH ~ 6.5, precipitation of metal hydroxides and of protein occurred. Fig. 3 presents the absorption spectra for free 5 mM NaC13 at pH 6.5, the spectrum resulting from introduction of enzyme to this metal ion solution as described in Methods, and the difference absorption spectrum obtained for comparison of these two solutions. In the difference spectrum, in the 300-600-nm region one observes peaks at 348, 355.5, 523, 577.5, 580, and 583.5 nm; and troughs are seen at 346.5, 351,354, 521,522, 571.5, and 573.5 nm. The spectrum for E + Nd, containing free and bound Nd(III), appears to have lost fine structure. However, this could result from a red shift for bound metal ions at the most sensitive bands. Such a shift has been attributed in spectroscopic model ligand work to a nephalauxetic effect, the tendency of a strong electrostatically negative ligand environment around the closedshell ion to expand the electron cloud of the metal, thus shifting its characteristic transitions to longer wavelengths [21, 26-28]. At this point it was also attempted to introduce a fluorescent rare earth ion probe, Tb(IlI), as had been done with Nd(III). Although Tb(III) was seen in Fig. 1 to activate the enzyme for catalysis, the fluorescence of the ion is not enhanced by binding to the protein upon excitation at 280 nm or at any of the major Tb(III)

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vs Nd(III). absorption wavelengths. This may result from lack of any aromatic chromophore, tryptophan or tyrosine sidechain groups, near the bound ion site to aid in energy transfer as was the case in concanavalin A [11], for example.

(5) Model studies In order to attempt to interpret the Nd(III) difference spectrum of Fig. 3, comparisons were made to similar spectra with various ligands. It has been a disappointment in this area that more definitive quantitative results with different model ligands have not been obtained to date. Nonetheless useful qualitative observations and correlations can be made at this point. Both A e and bandwidth have been found to be directly proportional to the strength of the complex formed, as reflected in the hypersensitive bands at 521 and 577 nm for Nd(III), and of the symmetry complex is also important. The extinction coefficient at 583 nm for enzyme-bound Nd(III) is calculated to be approx. 100 M -1 .cm -1. Also of importance is the general shape of the difference spectrum. In studies of carboxylic and amino acid model ligands by difference absorption spectroscopy, Birnbaum and Darnall [19] and Darnall, D. W. (personal communication), found that monodentate ligands (e.g. acetate) appear to enhance absorption in the 560-590-nm region, whereas polydentate ligands (e.g. EDTA, glutamate, serine, cysteine, and histidine) produce both a peak and a pronounced though in the 570-575-nm region. Polydentate ligands can elicit much stronger A e changes than monodentate ones. We have found that o-phenanthrolene or o,o'-bipyridyl ligands produced < 5 ~ enhancement of these hypersensitive bands. Thus, although nitrogen ligands can coordinate, they do so most effectively only when attached to carboxylate groups elsewhere in the molecule. Other examples of this effect may include EDTA complexes [29]. Based on such model studies, we infer that Nd(III) in the protein is strongly

438 bound in a polydentate manner, very likely to carboxylate sidechain groups. Interaction with serine - O H , cysteine - S H , or histidine -imidazole groups are also possible and likely, particularly since the fine structure seen in the difference spectrum in Fig. 3 is also observed mainly with model ligands such as these (Darnall, D. W., personal communication).

(6) Small molecule-induced spectral perturbations U p o n addition of each substrate in separate experiments to the Nd-enzyme complex (sample cuvette) and to free Nd(III) (reference cuvette), the difference spectrum is perturbed in different and unique manners, as is shown in Fig. 4. Bound ammonia alters the 570-575-nm through more than it does the 575-585-nm peaks or

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439 A rather wide variety of feedback modifiers of the enzyme were also added to the sample and reference cuvettes in separate experiments, the results of which are shown in Fig. 5. Virtually every response or change is different and each tends to weaken the intensity of the difference spectrum. The strongest effects are seen with glucosamine 6-phosphate (GN-6-P) and L-tryptophan, partial competitive inhibitors of ammonia and glutamine binding, respectively. L-Histidine, which inhibits NH3 binding, has a weaker effect, mainly on the 565-590-nm hypersensitive bands, as ~ e s glycine, a 1/modifier. Carbamyl phosphate and GDP act to increase fine structure somewhat but not to weaken band strength appreciably. The lack of effect by L-

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440 alanine is consistent with an observed dependence on bound substrate for its binding and much weaker effect on adenylylated than unadenylylated subunits. The unique nature of the responses in Fig. 5 serves to support the view that each modifier interacts in a unique manner with the enzyme, most likely with each binding at a separate site on the protein subunit. More detailed interpretation is not possible at present, although one may speculate that the symmetry of the ligand environment about the bound Nd 3+ is altered, rather than strength of the complex, in these latter spectra. DISCUSSION The general importance of the present data is to show that rare earth ions can be very useful as spectrophotometric probes in selected biological systems. With a system less restricted by solubility and aggregation problems than E. coli glutamine synthetase, one presumably could obtain small molecule binding constants by perturbation of the difference spectrum. This has been possible with pyruvate kinase and concanavalin A, to some extent [11, 12]. The potential use of these ionic chromophores in conjunction with activity assays and high frequency N M R shift studies [10, 30] is a very promising area for future study. The present results are unique in that this represents only the second enzyme, with a-amylase [8], which is activated rather than inhibited by bound rare earth ion. The presence of multiple types of metal binding sites on this enzyme, the tightest set of which (nl) is originally occupied by Mn(II) in these experiments may raise some question as to the exact site to which added Nd(III) becomes ultimately bound. It has been assumed here that this is the n2 set of sites. This view is supported by several facts: (a) Introduction by dialysis of Nd(III) to apoenzyme either before or simultaneously with Mn(II) leads to precipitation of protein, whereas introduction of Nd(III) after Mn(II) does not. (b) The Nd(III) titration curves in Fig. 2 show inflection points at or near nucleotide levels. (c) Addition of Nd(III) to the EI~-(Mn2+)~ 2 complex does not result in an increase in free Mn(II), as probed by EPR, or in displacement of bound Mn(II), as probed by pulsed N M R studies of water proton relaxation rates. Therefore, as a working hypothesis, we would presently favor the interpretation that under the conditions used and described in Methods and Fig. 3, Nd(III) binds preferentially at n2 sites and does not displace or exchange with Mn(I1) at the nl sites. Whether Nd(III) actually binds to nz sites as do Mg 2+ or Mn 2+ or in slightly displaced or rearranged positions remains to be established by more detailed experimentation. ACKNOWLEDGEMENTS This research was supported in part by grants from the National Science Foundation, G U 3182 to Rensselaer and GB 34751 to F. C. W. We wish to thank Dr Dennis Darnall for helpful discussions, encouragement with this work, and for making available unpublished results. We also thank Dr Darnall and the Kerr McGee Chemical Corporation for providing samples of rare earth oxides. Dr Ron Bailey is thanked for providing access to the atomic absorption instrument, Dr J. A. Fee for help with obtaining the EPR spectra, and Dr J. J. ViUafranca for help with the pulsed N M R experiments.

441 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

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