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)     

Subunit communication in tetrameric class 2 human liver aldehyde dehydrogenase as the basis for half-of-the-site reactivity and the dominance of the oriental subunit in a heterotetramer

Data has been published showing that in heterotetrameric liver mitochondrial aldehyde dehydrogenase composed of the active (E487) and the inactive Oriental-variant (K487) subunit, the Oriental variant was dominant and caused the inactivation of the E487 subunit. The published structures of the enzyme showed that the glutamate at position 487 is salt bonded to an arginine (475) in a different subunit. Arg475 was mutated to a glutamine to test for its importance in causing the Oriental variant to be an enzyme with a high Km for NAD and a low specific activity. Unexpectedly, the R475Q mutant exhibited positive cooperativity in NAD binding with a Hill coefficient of 2. Individual heterotetramers composed of subunits of E487 and K487 were produced by making changes to two residues on the surface of the enzyme and then co-expressing both cDNAs in E. coli. The E(3)K form had essentially 50% the activity of the E(4) homotetrameric form while EK(3) had essentially the same properties as did the homotetrameric K(4) Oriental variant. This showed that in a dimer pair composed of one K- and one E- subunit the K-subunit became dominant and caused the inactivation of its E-partner. Further, pre-steady state burst data and steady state kinetic data make it appear that there was one functioning active subunit in each of the dimer pairs that made up the tetrameric enzyme. Thus, the half-of-the-site reactivity is a result of having one functioning and one non-functioning subunit in each dimer pair. The actual structural basis for this is still not understood, but could be related to the E487-R475 inter-dimer salt bond.     

Subunit communication in tetrameric class 2 human liver aldehyde dehydrogenase as the basis for half-of-the-site reactivity and the dominance of the oriental subunit in a heterotetramer

Data has been published showing that in heterotetrameric liver mitochondrial aldehyde dehydrogenase composed of the active (E487) and the inactive Oriental-variant (K487) subunit, the Oriental variant was dominant and caused the inactivation of the E487 subunit. The published structures of the enzyme showed that the glutamate at position 487 is salt bonded to an arginine (475) in a different subunit. Arg475 was mutated to a glutamine to test for its importance in causing the Oriental variant to be an enzyme with a high Km for NAD and a low specific activity. Unexpectedly, the R475Q mutant exhibited positive cooperativity in NAD binding with a Hill coefficient of 2. Individual heterotetramers composed of subunits of E487 and K487 were produced by making changes to two residues on the surface of the enzyme and then co-expressing both cDNAs in E. coli. The E₃K form had essentially 50% the activity of the E₄ homotetrameric form while EK₃ had essentially the same properties as did the homotetrameric K₄ Oriental variant. This showed that in a dimer pair composed of one K- and one E- subunit the K-subunit became dominant and caused the inactivation of its E-partner. Further, pre-steady state burst data and steady state kinetic data make it appear that there was one functioning active subunit in each of the dimer pairs that made up the tetrameric enzyme. Thus, the half-of-the-site reactivity is a result of having one functioning and one non-functioning subunit in each dimer pair. The actual structural basis for this is still not understood, but could be related to the E487–R475 inter-dimer salt bond.     

The effect of valine substitution for glycine in the dimer interface of citrate synthase from Thermoplasma acidophilum on stability and activity

To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (K(m) for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., V(max), k(cat) and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 --> Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS.     

Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa

Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI(C321S) is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI(C321S), FXI(C321S,K331A), FXI(C321S,E287A), FXI(C321S,I290A), FXI(C321S,Y329A), FXI(C321S,L284A), FXI(C321S,K331R), and FXI(C321S,H343A) were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI(C321S) and FXI(C321S,H343A) existed in monomer/dimer equilibrium (K(d) approximately 40 nm), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K(d)=3-38 microm). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.     

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.     

Reversible dissociation/association of D-amino acid transaminase subunits: properties of isolated active dimers and inactive monomers

The crystal structure of dimeric D-amino acid transaminase shows that the two Trp-139 sites are located in a hydrophobic pocket at the interface between the subunits and that the two indole side chains face one another and are within 10 A of coenzyme. This enzyme prefers an aromatic character at position 139, as previously demonstrated by the finding that Phe-139 but no other substitution tested provides the maximum degree of thermostability and catalytic efficiency. Here we show that an equilibrium between active dimers and inactive monomers can be demonstrated with the W139F mutant enzyme, whereas with the wild-type enzyme the subunit interface is so tight that a study of this equilibrium is precluded. We show how the processes of dimerization of monomers and dissociation of dimers to monomers are controlled. Lower pH (5.0) favors monomer formation from dimers. Gel filtration and activity analysis show that at higher pH (7.0) the monomers combine to form active dimers with a K(d) of 0.17 microM. This assembly process is relatively slow and takes several hours for completion, thereby permitting accurate measurement of kinetics and equilibrium parameters. Absorption and circular dichroism spectra of dimers and monomers are significantly different, indicating that the environment around the cofactor is very likely altered between them. The circular dichroism peak of the W139F dimer at 418 nm is less negative than that of the wild-type enzyme in accordance with its lower visible absorbance; the circular dichroism peak of the W139F monomer at 418 nm is more negative than that of the wild-type enzyme. The dissociation of dimers to monomers has also been studied by taking advantage of these spectral differences, thus permitting the rates of the dissociation and the reassociation to be calculated and compared. 2-Mercaptoethanol assists in the conversion of monomers to dimers. The results here describe dissociation/reassociation in the dimeric enzyme under native conditions without denaturants.     

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3

Interactions between loops 2, 2′ and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment. Stability measurements show that only the K242A single mutant decreases stability of the dimer, whereas both single mutants and the double mutant demonstrate much lower activity compared to wild-type caspase-3. Structural studies of the caspase-3 variants show the involvement of K242 in hydrophobic interactions that stabilize helix 5, near the dimer interface, and the role of E246 appears to be to neutralize the positive charge of K242 within the hydrophobic cluster. Overall, the results suggest E246 and K242 are important in procaspase-3 for their interaction with neighboring residues, not with one another. Conversely, formation of the K242–E246 salt bridge in caspase-3 is needed for an accurate, stable conformation of loop L4 and proper active site formation in the mature enzyme.     

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3α-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 °C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3α-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.     

Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. A versatile tool to rate inhibitor effects?

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.     

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.     

Alcohol dehydrogenase isozymes of Triticum: Dissociation and recombination of subunits

Summary Conditions have been defined for the dissociation of active forms of Triticum alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1) into monomers and for the reassociation of the subunits into active enzymes. Results of experiments in which the subunits of genetically controlled electrophoretic variants of alcohol dehydrogenase were dissociated and recombined in crude tissue extracts support the hypothesis that the enzyme exists functionally as a dimer in Triticum.     

Characterization of heterodimeric alkaline phosphatases from Escherichia coli: an investigation of intragenic complementation

Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant versions of alkaline phosphatase are combined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phosphate K(m) were observed for certain combinations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.     

Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants

Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid. A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2). Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) [Koyama, T., et al. (1995) Biochem. Biophys. Res. Commun. 212, 681-686], respectively. These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit.     

Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.