Mol Of Dimer Research Articles

Entropies of Organolithium Aggregation Based on Measured Microsolvation Numbers

The recent measurement (J. Am. Chem. Soc.2008, 130, 14179–14188) of the microsolvation numbers of monodentate, nonchelating ethereal donor ligands coordinating to the monomers and dimers of two sterically shielded ═C(aryl)–Li compounds permits the determination of well-founded dimerization enthalpies (ΔH0) and entropies (ΔS0) from properly formulated equilibrium constants, which must include the concentrations of the free donor ligands. The monomers are found to dimerize endothermically (ΔH0 > 0) in [D8]toluene solution in the presence of the donor tBuOMe or THF, but only slightly exothermically (ΔH0 = −0.5 kcal per mol of dimer) with the donor Et2O. The dimerization entropies ΔS0 (in cal mol–1 K–1) with the respective equivalents of released donor ligands are 7.2 and 11.0 (with 2 equiv of tBuOMe in the two cases), 6.1 (with 2 Et2O), and 34.1 (with 4 THF). It is shown that the improper omission of microsolvation from the equilibrium constant (a usual practice when the ligand numbers are not known) can lea...

Mua (HP0868) Is a Nickel-Binding Protein That Modulates Urease Activity in Helicobacter pylori

A novel mechanism aimed at controlling urease expression in Helicobacter pylori in the presence of ample nickel is described. Higher urease activities were observed in an hp0868 mutant (than in the wild type) in cells supplemented with nickel, suggesting that the HP0868 protein (herein named Mua for modulator of urease activity) represses urease activity when nickel concentrations are ample. The increase in urease activity in the Δmua mutant was linked to an increase in urease transcription and synthesis, as shown by quantitative real-time PCR, SDS-PAGE, and immunoblotting against UreAB. Increased urease synthesis was also detected in a Δmua ΔnikR double mutant strain. The Δmua mutant was more sensitive to nickel toxicity but more resistant to acid challenge than was the wild-type strain. Pure Mua protein binds 2 moles of Ni2+ per mole of dimer. Electrophoretic mobility shift assays did not reveal any binding of Mua to the ureA promoter or other selected promoters (nikR, arsRS, 5′ ureB-sRNAp). Previous yeast two-hybrid studies indicated that Mua and RpoD may interact; however, only a weak interaction was detected via cross-linking with pure components and this could not be verified by another approach. There was no significant difference in the intracellular nickel level between wild-type and mua mutant cells. Taken together, our results suggest the HP0868 gene product represses urease transcription when nickel levels are high through an as-yet-uncharacterized mechanism, thus counterbalancing the well-described NikR-mediated activation.

Mapping the Folding Free Energy Surface for Metal-free Human Cu,Zn Superoxide Dismutase

Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is − 20.37( ± 1.04) kcal (mol dimer) − 1 . A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

Backbone-backbone hydrogen-bonding interactions are a ubiquitous and highly conserved structural feature of proteins that adopt the same fold (i.e., have the same overall backbone topology). This work addresses the question of whether or not this structural conservation is also reflected as a thermodynamic conservation. Reported here is a comparative thermodynamic analysis of backbone hydrogen bonds in two proteins that adopt the same fold but are unrelated at the primary amino acid sequence level. With amide-to-ester bond mutations introduced by total chemical synthesis methods, the thermodynamic consequences of backbone-backbone hydrogen-bond deletions at five different structurally equivalent positions throughout the beta-alpha-alpha fold of Arc repressor and CopG were assessed. The ester bond-containing analogues all folded into native-like three-dimensional structures that were destabilized from 2.5 to 6.0 kcal/(mol dimer) compared with wild-type controls. Remarkably, the five paired analogues with amide-to-ester bond mutations at structurally equivalent positions were destabilized to exactly the same degree, regardless of the degree to which the mutation site was buried in the structure. The results are interpreted as evidence that the thermodynamics of backbone-backbone hydrogen-bonding interactions in a protein fold are conserved.

Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: cloning, purification, and characterization of 2,6-dihydroxypyridine 3-hydroxylase.

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four α-helix bundle protein

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-α-helix motif of the wild-type structure is given up. Instead a homotetrameric four-α-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71°C characteristic of the wild-type protein to 101°C for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer) −1 to 195.1 kJ (mol of tetramer) −1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of Δ H=1073±30 kJ (mol of tetramer) −1 and a heat capacity change at the transition temperature of Δ N D C p=14.9(±)3% kJ (mol of tetramer × K) −1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van’t Hoff enthalpy values.

Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction.

Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-alpha-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric alpha helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101 degreesC. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k2 varies dramatically with both temperature and denaturant concentration. At 25 degreesC and 6.5 M GdnHCl concentration k2 is 4200 L.(mol of dimer)-1.s-1, whereas at 4.4 M GdnHCl a value of k2 = 0.9 L.(mol of dimer)-1.s-1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from DeltaH# = 29.1 kJ.mol-1 at 6. 5 M GdnHCl to Delta H# = 79.7 kJ.mol-1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings.

CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein.

Biological sensing of small molecules such as NO, O2, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium Rhodospirillum rubrum responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. In this study we report the purification of wild-type CooA from its native organism, R. rubrum, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein's response to CO is proposed.

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.

An Unusual Dehalogenating Peroxidase from the Marine Terebellid Polychaete Amphitrite ornata

The terebellid polychaete Amphitrite ornata produces no detectable volatile halogenated secondary metabolites, but frequently inhabits coastal marine sediments heavily contaminated with anthropogenic or biogenic haloaromatic compounds. This animal contains high levels of two very unusual enzymes, dehalogenating peroxidases. We have purified and partially characterized one of these dehaloperoxidases, DHP I. DHP I is a heme enzyme (Mr = 30,790) composed of two identical subunits (Mr = 15,529) and is very rich in the amino acids aspartic acid (+ asparagine) and glutamic acid (+ glutamine). The enzyme converts trihalogenated phenols, such as 2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for this reaction is 5.0. DHP I is also active against di- and monohalogenated phenols and will oxidize bromo-, chloro-, and fluorophenols. We have identified similar dehaloperoxidase activities in other infaunal polychaetes, including halometabolite-producing species.

Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy.

Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

Effects of cavity‐creating mutations on conformational stability and structure of the dimeric 4‐α‐helical protein ROP: Thermal unfolding studies

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41-->Val and Leu-41-->Ala) in the dimeric 4-alpha-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in alpha-helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. delta H zero values at the respective transition temperatures, T 1/2 (71.0, 65.3, and 52.9 degrees C at 0.5 mg/ml) decrease from 580 +/- 20 to 461 +/- 20 kJ/(mol of dimer) and 335 +/- 20 kJ/(mol of dimer) for wild-type ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L41V, and L41A, respectively. The conformational stabilities at 25 degrees C expressed by the standard Gibbs energies of denaturation, delta GzeroD, are 71.7, 61.1, and 46.1 kJ/(mol of dimer). The corresponding transition enthalpies have been obtained from extrapolation using the cDp(T) and cNp(T) functions. Their values at 25 degrees C are 176.3, 101.9, and 141.7 kJ/(mol of dimer) for wild-type ROP, L41V, and L41A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH2 group, the average delta delta GzeroD value is -5.0 +/- 1 kJ/(mol of CH2 group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu-->Val and Leu-->Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state.

A Thermodynamic Study of Mutant Forms ofStreptomycesSubtilisin Inhibitor. II. Replacements at the Interface of Dimer Formation, Val13

Amino acid replacements in a homodimeric protein,Streptomycessubtilisin inhibitor, at the position of Val13, which is located at the center of the β-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21 °C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)−1for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy, to 0.84 kcal (mol dimer)−1for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)−1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2⇌ 2D, which was the case for the Leu and Ile mutants as well as for the wild-type, to N2⇌ 2N*⇌ 2D, where the first step can be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol−1for the Ala mutant at 66.95 °C at pH 9.5 and lower for the other three mutants.

The thermodynamics of the unfolding of an isolated protein subdomain The 255–316 C-terminal fragment of thermolysin

Differential scanning calorimetry has been used to study the thermal unfolding of the 255–316 C-terminal fragment of thermolysin. The concentration effect on the calorimetric transitions of the fragment in 0.1 M NaCl and 20 mM phosphate buffer, pH 7.5, shows that it behaves as a highly stable dimer in solution, whithin the concentration range 0.19–4.55 mg/ml, undergoing a reversible two-state thermal unfolding process. The thermodynamic parameters of unfolding (Δ G = 60 ± 6 kJ/(mol of dimer) at 20°C) are similar to those normally observed for small, compact, globular proteins. This and previous studies [1989, Eur. J. Biochem. 180, 513–518] show that the 255–316 fragment folds into a stable, native-like globular structure.

Smooth muscle calponin-caltropin interaction: effect on biological activity and stability of calponin.

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)

What is the global minimum energy structure of the water hexamer? The importance of nonadditive interactions

The global minimum energy structures of the water hexamer predicted by widely used analytic water potentials are very different from each other, while the cyclic hexamer does not appear to be a low-lying energy structure. However, high levels of ab initio calculation predict that a number of low-lying energy conformers including the cyclic conformer are almost all isoenergetic due to the balance of two-body and nonadditive interactions. For modeling of water potentials, we suggest that the binding energy of the dimer be between −5.0 and −4.7 kcal (mol dimer)−1, while the three-body corrections be taken into account to a large extent.

Subunit interactions provide a significant contribution to the stability of the dimeric four-alpha-helical-bundle protein ROP.

Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP.

Iron- and manganese-containing superoxide dismutases from Methylomonas J: identity of the protein moiety and amino acid sequence

Mn-superoxide dismutase (SOD) and Fe-SOD were isolated from Methylomonas J, an aerobic methylotrophic bacterium, grown in methylamine media containing either manganese (Mn-rich medium) or iron (Fe-rich medium), respectively. The specific activity of the Mn-SOD was 2250 units mg-1 (mol of Mn)-1 (mol of dimer)-1, and the metal content of the enzyme was 0.98 mol of Mn and 0.12 mol of Fe per mole of dimer, while those of Fe-SOD were 88.5 units mg-1 (mol of Fe)-1 (mol of dimer)-1 and 1.04 mol of Fe and 0.02 mol of Mn. The electrophoretic mobilities in the presence of sodium dodecyl sulfate, with or without urea, and the chromatographic behavior on an HPLC column using an octadodecyl silicated column and a gel permeation column were identical. Amino acid compositions were practically indistinguishable in both SODs. The enzyme activity was restored by dialysis of an apoprotein obtained from the Mn-enzyme with either manganese sulfate or ferrous ammonium sulfate up to an activity level similar to that for the native Mn-SOD and the native Fe-SOD, respectively. The same result has been reported with the reconstitution using an apoprotein obtained from the Fe-enzyme [Yamakura, F., Matsumoto, T., & Terauchi, K. (1990) Free Radical Res. Commun. (in press)]. These results suggest the possibility that both types of SODs are composed of a single apoprotein synthesized in cells grown in either the Fe-rich medium or the Mn-rich medium.(ABSTRACT TRUNCATED AT 250 WORDS)

L-Histidinol dehydrogenase, a Zn 2+-metalloenzyme

The enzymatic activity of l-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 m m MnCl 2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn 2+/mol dimer, K D = 37 μM. ZnCl 2, which prevented the MnCl 2 stimulation, also bound to the enzyme, 1.2 g-atom/ mol dimer, K D = 51 μM, and prevented Mn 2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 m urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl 2 or ZnCl 2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl 2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl 2 or ZnCl 2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn 2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn 2+, but which may bind Mn 2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn 2+ fulfills an essential structural and/or catalytic role.

Asymmetry of tyrosyl-tRNA synthetase in solution.

The tyrosyl-tRNA synthetase from Bacillus stearothermophilus crystallizes as a symmetrical dimer with each subunit having a complete active site. The enzyme-substrate complexes, however, are known to be asymmetrical in solution because the enzyme exhibits half-of-the-sites activity by binding tightly only 1 mol of tyrosine or 1 mol of tyrosyl adenylate per mole of dimer. Evidence is now presented that the unligated enzyme is also asymmetrical in solution. Symmetry was investigated by construction of heterodimers containing one full-length subunit and one truncated subunit, allowing the introduction of different mutations into each monomer. Each dimer is active at only one site, but the site used is randomly distributed between the subunits. Each heterodimer thus consists of two equal populations, one activating tyrosine at a full-length subunit and the other at the truncated subunit. No detectable interconversion is found between active and inactive sites over several minutes either in the absence of substrates or when the enzyme is turning over in the steady state. Kinetic evidence implies that wild-type enzyme is inherently asymmetrical even in the absence of substrate.