Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.     

Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces

The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25 degrees C the tetramer-dimer dissociation constants for each single-mutant enzyme are similar, about 10(-6) M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramer-monomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10(-15) M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10(-28) M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.     

Human aldolase A of a hemolytic anemia patient with Asp-128----Gly substitution: characteristics of an enzyme generated in E. coli transfected with the expression plasmid pHAAD128G

Aldolase A derived from a hemolytic anemia patient with aldolase A deficiency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme [Kishi et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8623-8627]. We constructed an Escherichia coli expression plasmid, pHAAD128G, which carries the mutant aldolase A [aldolase A(D-G)] cDNA, and the enzyme generated in E. coli transfected with the expression plasmid was purified and characterized. Conversion of Asp to Gly at the 128th position in the enzyme rendered the enzyme thermolabile and susceptible to tryptic digestion. CD spectra analysis also revealed that the mutant enzyme had a remarkable conformation change with a decrease of regular form in the molecule. Addition of glycerol or some other polyalcohols during thermal treatment protected this altered enzyme (but not the normal enzyme) against denaturation and activity decrease. In order to determine the function of the amino acid residue at the 128th position, two artificial mutant enzymes with the substitutions of Glu for Asp [aldolase A(D-E)] and Ser for Asp [aldolase A(D-S)], respectively, at the position were constructed by site-directed mutagenesis and characterized. These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Importance of a conserved residue, aspartate-162, for the function of Escherichia coli aspartate transcarbamoylase.

Aspartate-162 in the catalytic chain of aspartate transcarbamoylase is conserved in all of the sequences determined to date. The X-ray structure of the Escherichia coli enzyme indicates that this residue is located in a loop region (160's loop) that is near the interface between two catalytic trimers and is also close to the active site. In order to test whether this conserved residue is important for support of the internal architecture of the enzyme and/or involved in transmitting homotropic and heterotropic effects, the function of this residue was studied using a mutant version of the enzyme with an alanine at this position (Asp-162----Ala) created by site-specific mutagenesis. The Asp-162----Ala enzyme exhibits a 400-fold reduction in the maximal observed specific activity, approximately 2-fold and 10-fold decreases in the aspartate and carbamoyl phosphate concentrations at half the maximal observed specific activity respectively, a loss of homotropic cooperativity, and loss of response to the regulatory nucleotides ATP and CTP. Furthermore, equilibrium binding studies indicate that the affinity of the mutant enzyme for CTP is reduced more than 10-fold. The isolated catalytic subunit exhibits a 660-fold reduction in maximal observed specific activity compared to the wild-type catalytic subunit. The Km values for aspartate and carbamoyl phosphate for the Asp-162----Ala catalytic subunit were within 2-fold of the values observed for the wild-type catalytic subunit. Computer simulations of the energy-minimized mutant enzyme indicate that the space once occupied by the side chain of Asp-162 may be filled by other side chains, suggesting that Asp-162 is important for stabilizing the internal architecture of the wild-type enzyme.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.     

Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.

A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.     

Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution

Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 A away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.     

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit-subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis. Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids. Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment. The mutations M256V and M256A increased thermostability by 2.0 and 6.0 degrees C, respectively, whereas the mutations M256L and M256I had no effect. Thermostability of the M256F mutated enzyme was 4.0 degrees C lower than that of the wild-type enzyme. To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability. The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256. Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded. These results suggest that Met256 prevents favorable packing. Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH. The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit-subunit interface and is not always optimized in the mesophilic IPMDH enzyme.     

Dynamics-function correlation in Cu, Zn superoxide dismutase: a spectroscopic and molecular dynamics simulation study

A single mutation (Val29-->Gly) at the subunit interface of a Cu, Zn superoxide dismutase dimer leads to a twofold increase in the second order catalytic rate, when compared to the native enzyme, without causing any modification of the structure or the electric field distribution. To check the role of dynamic processes in this catalytic enhancement, the flexibility of the dimeric protein at the subunit interface region has been probed by the phosphorescence and fluorescence properties of the unique tryptophan residue. Multiple spectroscopic data indicate that Trp83 experiences a very similar, and relatively hydrophobic, environment in both wild-type and mutant protein, whereas its mobility is distinctly more restrained in the latter. Molecular dynamics simulation confirms this result, and provides, at the molecular level, details of the dynamic change felt by tryptophan. Moreover, the simulation shows that the loops surrounding the active site are more flexible in the mutant than in the native enzyme, making the copper more accessible to the incoming substrate, and being thus responsible for the catalytic rate enhancement. Evidence for increased, dynamic copper accessibility also comes from faster copper removal in the mutant by a metal chelator. These results indicate that differences in dynamic, rather than structural, features of the two enzymes are responsible for the observed functional change.     

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.     

Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.     

Effects of engineering complementary charged residues into the hydrophobic subunit interface of tyrosyl-tRNA synthetase. Appendix: Kinetic analysis of dimeric enzymes that reversibly dissociate into inactive subunits

Wild-type tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is a symmetrical dimer. Four different heterodimeric enzymes have been produced by site-directed mutagenesis at the subunit interface so that the monomers are linked by a potential salt bridge in a hydrophobic environment. The two Phe-164 residues of wild-type TyrTS are on the axis of symmetry and interact in a hydrophobic region of the subunit interface. Mutation of Phe-164 to aspartate or glutamate in full-length TyrTS and to lysine or arginine in an active truncated enzyme (delta TyrTS) induces reversible dissociation of the enzyme into inactive monomers. Mixing mutants in equimolar amounts produces four different heterodimers: TyrTS(Asp-164)-delta TyrTS(Lys-164); TyrTS(Asp-164)-delta TyrTS(Arg-164); TyrTS(Glu-164)-delta TyrTS(Lys-164); TyrTS(Glu-164)-delta TyrTS(Arg-164). A general method is derived for analyzing the kinetics of dimeric enzymes that reversibly dissociate into inactive subunits. Application to mutants of TyrTS allows estimation of dissociation constants (Kd values) for the dimers. At pH 7.8, the heterodimers have Kd values of 6-14 microM, whereas for homodimers Kd = 120-4000 microM. These values decrease to about 30 microM for homodimers of TyrTS(Asp-164), TyrTS(Glu-164), and delta TyrTS(Lys-164) when the pH favors uncharged forms of the side chains at position 164. Each of the four salt bridges engineered into the hydrophobic subunit interface of TyrTS appears, therefore, to be weak. These engineered salt bridges may be compared with naturally occurring ones. In the latter, there are complementary interactions between the charges in the salt bridge with polar groups in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer–dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1 Å) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.     

Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.     

Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytically active monomer of glutathione S-transferase pi and key residues involved in the electrostatic interaction between subunits

Human glutathione transferase pi (GST pi) has been crystallized as a homodimer, with a subunit molecular mass of approximately 23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST pi, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K(m) for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K(m)(GSH), whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V(max). The GST pi retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.     

Brownian dynamics simulations of glycolytic enzyme subsets with F-actin

Previous Brownian dynamics (BD) simulations identified specific basic residues on fructose-1,6-bisphophate aldolase (aldolase) (I. V. Ouporov et al., Biophysical Journal, 1999, Vol. 76, pp. 17-27) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I. V. Ouporov et al., Journal of Molecular Recognition, 2001, Vol. 14, pp. 29-41) involved in binding F-actin, and suggested that the quaternary structure of the enzymes may be important. Herein, BD simulations of F-actin binding by enzyme dimers or peptides matching particular sequences of the enzyme and the intact enzyme triose phosphate isomerase (TIM) are compared. BD confirms the experimental observation that TIM has little affinity for F-actin. For aldolase, the critical residues identified by BD are found in surface grooves, formed by subunits A/D and B/C, where they face like residues of the neighboring subunit enhancing their electrostatic potentials. BD simulations between F-actin and aldolase A/D dimers give results similar to the native tetramer. Aldolase A/B dimers form complexes involving residues that are buried in the native structure and are energetically weaker; these results support the importance of quaternary structure for aldolase. GAPDH, however, placed the critical residues on the corners of the tetramer so there is no enhancement of the electrostatic potential between the subunits. Simulations using GAPDH dimers composed of either S/H or G/H subunits show reduced binding energetics compared to the tetramer, but for both dimers, the sets of residues involved in binding are similar to those found for the native tetramer. BD simulations using either aldolase or GAPDH peptides that bind F-actin experimentally show complex formation. The GAPDH peptide bound to the same F-actin domain as did the intact tetramer; however, unlike the tetramer, the aldolase peptide lacked specificity for binding a single F-actin domain.