A highly specific monomeric isocitrate dehydrogenase from Corynebacterium glutamicum

The monomeric isocitrate dehydrogenase (IDH) of Corynebacterium glutamicum is compared to the topologically distinct dimeric IDH of Escherichia coli. Both IDHs have evolved to efficiently catalyze identical reactions with similar pH optimum as well as striking specificity toward NADP and isocitrate. However, the monomeric IDH is 10-fold more active (calculated as kcat/Km.isocitrate/Km.NADP) and 7-fold more NADP-specific than the dimeric enzyme, favoring NADP over NAD by a factor of 50,000. Such an extraordinary coenzyme specificity is not rivaled by any other characterized dehydrogenases. In addition, the monomeric enzyme is 10-fold more specific for isocitrate. The spectacular substrate specificity may be predominantly attributed to the isocitrate-assisted stabilization of catalytic complex during hydride transfer. No significant overall sequence identity is found between the monomeric and dimeric enzymes. However, structure-based alignment leads to the identification of three regions in the monomeric enzyme that match closely the three motifs located in the central region of dimeric IDHs and the homologous isopropylmalate dehydrogenases. The role of Lys253 as catalytic residue has been demonstrated by site-directed mutagenesis. Our results suggest that monomeric and dimeric forms of IDHs are functionally and structurally homologous.     

Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase

To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.     

Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.     

Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.     

Purification and activity of two phospholipase enzymes from Naja nigricolis nigricolis Reinhardt venom

Two phospholipase enzymes NN1 and NN2 were purified from the venom of Naja nigricolis nigricolis Reinhardt to apparent homogeneity. NN1 was purified by a two-step anion-exchange chromatography on DEAE-cellulose column while NN2 was purified by a combination of anion-exchange chromatography and gel filtration on Sephadex G-150. The enzyme NN1 moved homogenously on acrylamide gel as a monomer with a molecular weight of 65 kDa while NN2 was a dimer of 71 kDa. Both enzymes were clearly separated. Both enzymes hydrolyzed L-alpha-phosphatidyl choline with activities of 345.5 for NN1 and 727.8 micromol min(-1) x mg(-1) for NN2. The dimeric 71-kDa enzyme has a higher haemolytic and anticoagulant activity than the monomeric 65-kDa enzyme. It is apparent that the dimeric enzyme has a more pronounced activity than the monomer has, thus toxic activity may be related to the hydrolysis of phospholipids.     

Functional characterization of the mitochondrial cytochrome b-c1 complex: steady-state kinetics of the monomeric and dimeric forms

The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.     

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

Functional equivalence of monomeric (shark) and dimeric (bovine) cytochrome c oxidase

Cytochrome c oxidase isolated from hammerhead shark red muscle is monomeric in relation to the dimeric form of isolated bovine cytochrome c oxidase but in other ways bears a close resemblance to the enzyme isolated from mammalian tissue [1, 2]. Comparative studies of shark and bovine cytochrome c oxidase were extended to address the degree of functional similarity between the monomeric (shark) and dimeric (bovine) enzymes in the kinetics of peroxide binding and in the extent to which the catalytic action of the enzymes in vesicles can establish a proton gradient. Although the kinetics of peroxide binding and the proton pumping processes are complex, the dimeric and monomeric forms are quite similar with respect to these functional attributes. The kinetic heterogeneity of the process of peroxide binding is expressed in the shark enzyme as well as in the bovine enzyme, and both types of enzymes in vesicles can generate transmembrane proton gradients. On this basis we conclude that the dimeric state of isolated cytochrome c oxidase from mammalian sources is not essential for its function in vitro.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Human hemi-myeloperoxidase. Initial chlorinating activity at neutral pH, compound II and III formation, and stability towards hypochlorous acid and high temperature.

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two promoters, 'hemi-myeloperoxidase'. We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of H2O2 for complete conversion. Hemi-MPO was less stable at a high temperature (80 degrees C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (IC50 = 32 microM HOCl) compared to dimeric MPO (IC50 = 50 microM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.     

The monomeric dUTPase from Epstein-Barr virus mimics trimeric dUTPases

Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes cleaving dUTP into dUMP and pyrophosphate. They occur as monomeric, dimeric, or trimeric molecules. The trimeric and monomeric enzymes both contain the same five characteristic sequence motifs but in a different order, whereas the dimeric enzymes are not homologous. Monomeric dUTPases only occur in herpesviruses, such as Epstein-Barr virus (EBV). Here, we describe the crystal structures of EBV dUTPase in complex with the product dUMP and a substrate analog alpha,beta-imino-dUTP. The molecule consists of three domains forming one active site that has a structure extremely similar to one of the three active sites of trimeric dUTPases. The three domains functionally correspond to the subunits of the trimeric form. Domains I and II have the dUTPase fold, but they differ considerably in the regions that are not involved in the formation of the unique active site, whereas domain III has only little secondary structure.     

Evidence for the activity of immobilised monomers of triose phosphate isomerase.

Chicken muscle triose phosphate isomerase was immobilised by attachment to Sepharose 4B. The immobilised dimeric enzyme was dissociated with guanidinium chloride to yield bound monomeric triose phosphate isomerase. This regained activity on removal of the denaturant, showing that isolated monomers possess activity; the apparent K_m of the immobilished subunits was the same as that of the immobilised dimers. Under appropriate conditions, it was possible to rehybridise the immobilised monomers to native dimers, and also to form a hybrid dimer from the chicken muscle and rabbit muscle enzymes.     

Multifunctionality of lipoamide dehydrogenase: activities of chemically trapped monomeric and dimeric enzymes

Lipoamide dehydrogenase from pig heart exists in monomer-dimer equilibrium. The effect of the state of subunit aggregation on the multifunctionality of lipoamide dehydrogenase was investigated by the use of chemically trapped monomeric and dimeric enzymes. Reductive carboxymethylation with 2-mercaptoethanol-iodoacetate yields the stable monomeric enzyme which has been isolated for structural and kinetic studies. The chemically induced monomerization is accompanied by conformational changes resulting in an increased mobility of flavin-adenine dinucleotide. The chemically trapped monomer shows an enhanced diaphorase activity, a reduced electron transferase activity, and a complete loss in dehydrogenase as well as transhydrogenase activities. The enhanced diaphorase activity is associated with increased catalytic efficiencies and the reversal of an inhibitory NADH effect at high concentrations. Treatment of lipoamide dehydrogenase with dimethyl suberimidate gives amidinated samples containing crosslinked dimer. The crosslinked enzyme exhibits a higher dehydrogenase catalytic efficiency than the noncrosslinked enzyme with different kinetic mechanisms without significantly affecting the kinetic parameters of diaphorase reaction. Although the dimeric structure is intimately associated with the dehydrogenase activity, it does not preclude the diaphorase activity. An altered flavin-adenine dinucleotide environment accompanying monomerization is likely responsible for the enhanced diaphorase activity.     

Evidence for active intermediates during the reconstitution of yeast phosphoglycerate mutase

The reconstitution of the tetrameric phosphoglycerate mutase from bakers' yeast after denaturation in guanidine hydrochloride has been studied. When assays are performed in the presence of trypsin, it is found that reactivation parallels the regain of tetrameric structure. However, in the absence of trypsin, the regain of activity is more rapid, suggesting that monomeric and dimeric intermediates possess partial activity (35% of the value of native enzyme) which is sensitive to trypsin. When reconstitution is studied in the presence of substrates, it is again found that monomeric and dimeric intermediates possess 35% activity. Under these latter conditions, the activity of the monomer but not of the dimer is sensitive to trypsin.     

The chemical basis for interfacial activation of monomeric phospholipases A2. Autocatalytic derivatization of the enzyme by acyl transfer from substrate

A basic monomeric phospholipase A2 from the venom of the American water moccasin, Agkistrodon piscivorus piscivorus, undergoes Ca2+-dependent, autocatalytic acylation during the course of hydrolysis of both model and natural phospholipid substrates. Acylation occurs at 2 lysine residues, Lys-7 and Lys-10, in the NH2-terminal alpha-helical segment of the enzyme, and when both positions are fully derivatized, the stable bisacylphospholipase A2 becomes a dimer in solution. The acylated enzyme is fully activated toward monomolecular layers of lecithins. Similar studies applied to the monomeric phospholipases A2 from porcine pancreas and from the venom of Agkistrodon contortrix contortrix also showed irreversible activation of the enzymes by substrate with the same kinetic consequences and formation of dimers. Acylation thus enables these enzymes to overcome the lag period observed under such conditions with native monomeric phospholipases, a phenomenon referred to as interfacial activation. Activation of the enzyme by acylation potentiates the phospholipase for interfacial recognition via formation of a dimeric enzyme. The naturally occurring phospholipase A2 dimer from Crotalus atrox venom displays no lag in the hydrolysis of lecithin monolayers nor does it undergo substrate level acylation. These facts support our proposal that dimerization concomitant with acylation is responsible for the large rate enhancements seen in the hydrolysis of aggregated phospholipids by monomeric phospholipases. Our findings demonstrate for the first time a chemical mechanism for interfacial activation of and interfacial recognition by phospholipases A2.     

Purification and characterization of monomeric isocitrate dehydrogenase with NADP(+)-specificity from Vibrio parahaemolyticus Y-4.

NADP(+)-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pI of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to IDH-II and -I of Vibrio ABE-1) showed an immunochemical distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% beta-pleated sheet in the native state.     

Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells

Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase, azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

Kinetic pathways of formation and dissociation of the glycerol-3-phosphate dehydrogenase-fructose-1,6-bisphosphate aldolase complex.

Quantitative analysis of the time courses of fluorescence anisotropy changes due to the binding of fructose-1,6-bisphosphate aldolase to the dissociable cytoplasmic glycerol-3-phosphate dehydrogenase covalently labelled with fluorescent dye was carried out. The behaviour of the aldolase-dehydrogenase system seems to be consistent with a cyclic reversible model characterized by the formation and dissociation of complexes of both the monomeric and the dimeric forms of dehydrogenase with aldolase, and rapid equilibrium between the free monomeric and dimeric forms of dehydrogenase. The half-life time of the formation of dimeric dehydrogenase-aldolase complex at the concentration of the enzymes expected to exist in the cell (i.e. in the micromolar range) is some minutes, and the time needed for equilibration between the aldolase-bound dimeric and monomeric forms of dehydrogenase is a few minutes as well. Consequently, one may expect that both the formation and the dissociation of this heterologous enzyme complex have physiological relevance.