A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis

The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.     

Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase beta subunit in the energy coupling mechanism of the Na+ pump.

The membrane-bound beta subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the beta subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild-type enzyme and the beta subunit mutants N373D, N373L, S382A, S382D, S382T, R389A, and R389D. In these studies, pH profiles, Na+ binding affinities, Hill coefficients, Vmax values and inhibition by Na+ was determined. A prominent result is the complete lack of oxaloacetate decarboxylase activity of the S382A mutant at Na+ concentrations up to 20 mm and recovery of significant activities at elevated Na+ concentrations (KNa approximately 400 mm at pH 6.0), where the wild-type enzyme is almost completely inhibited. These results indicate impaired Na+ binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na+ concentrations is uncoupled from the vectorial events of Na+ or H+ translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism, which includes Na+ binding to center I contributed by D203 (region IIIa) and N373 (helix VIII) and center II contributed by Y229 (helix IV) and S382 (helix VIII). These centers are exposed to the cytoplasmic surface in the carboxybiotin-bound state of the beta subunit and become exposed to the periplasmic surface after decarboxylation of this compound. During the countertransport of 2 Na+ and 1 H+ Y229 of center II switches between the protonated and deprotonated Na+-bound state.     

Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.     

Mechanisms of sodium transport in bacteria

In some bacteria, an Na+ circuit is an important link between exergonic and endergonic membrane reactions. The physiological importance of Na+ ion cycling is described in detail for three different bacteria. Klebsiella pneumoniae fermenting citrate pumps Na+ outwards by oxaloacetate decarboxylase and uses the Na+ ion gradient thus established for citrate uptake. Another possible function of the Na+ gradient may be to drive the endergonic reduction of NAD+ with ubiquinol as electron donor. In Vibrio alginolyticus, an Na+ gradient is established by the NADH: ubiquinone oxidoreductase segment of the respiratory chain; the Na+ gradient drives solute uptake, flagellar motion and possibly ATP synthesis. In Propionigenium modestum, ATP biosynthesis is entirely dependent on the Na+ ion gradient established upon decarboxylation of methylmalonyl-CoA. The three Na(+)-translocating enzymes, oxaloacetate decarboxylase of Klebsiella pneumoniae, NADH: ubiquinone oxidoreductase of Vibrio alginolyticus and ATPase (F1F0) of Propionigenium modestum have been isolated and studied with respect to structure and function. Oxaloacetate decarboxylase consists of a peripheral subunit (alpha), that catalyses the carboxyltransfer from oxaloacetate to enzyme-bound biotin. The subunits beta and gamma are firmly embedded in the membrane and catalyse the decarboxylation of the carboxybiotin enzyme, coupled to Na+ transport. A two-step mechanism has also been demonstrated for the respiratory Na+ pump. Semiquinone radicals are first formed with the electrons from NADH; subsequently, these radicals dismutate in an Na(+)-dependent reaction to quinone and quinol. The ATPase of P. modestum is closely related in its structure to the F1F0 ATPase of E. coli, but uses Na+ as the coupling ion. A specific role of protons in the ATP synthesis mechanism is therefore excluded.     

Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na(+) pump

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.     

Crystal structure of the carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl-coenzyme A decarboxylase

Glutaconyl-CoA decarboxylase is a biotin-dependent ion pump whereby the free energy of the glutaconyl-CoA decarboxylation to crotonyl-CoA drives the electrogenic transport of sodium ions from the cytoplasm into the periplasm. Here we present the crystal structure of the decarboxylase subunit (Gcdalpha) from Acidaminococcus fermentans and its complex with glutaconyl-CoA. The active sites of the dimeric Gcdalpha lie at the two interfaces between the mono mers, whereas the N-terminal domain provides the glutaconyl-CoA-binding site and the C-terminal domain binds the biotinyllysine moiety. The Gcdalpha catalyses the transfer of carbon dioxide from glutaconyl-CoA to a biotin carrier (Gcdgamma) that subsequently is decarboxylated by the carboxybiotin decarboxylation site within the actual Na(+) pump (Gcdbeta). The analysis of the active site lead to a novel mechanism for the biotin-dependent carboxy transfer whereby biotin acts as general acid. Furthermore, we propose a holoenzyme assembly in which the water-filled central channel of the Gcdalpha dimer lies co-axial with the ion channel (Gcdbeta). The central channel is blocked by arginines against passage of sodium ions which might enter the central channel through two side channels.     

Sodium ion-translocating decarboxylases

The review is concerned with three Na(+)-dependent biotin-containing decarboxylases, which catalyse the substitution of CO(2) by H(+) with retention of configuration (DeltaG degrees '=-30 kJ/mol): oxaloacetate decarboxylase from enterobacteria, methylmalonyl-CoA decarboxylase from Veillonella parvula and Propiogenium modestum, and glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. The enzymes represent complexes of four functional domains or subunits, a carboxytransferase, a mobile alanine- and proline-rich biotin carrier, a 9-11 membrane-spanning helix-containing Na(+)-dependent carboxybiotin decarboxylase and a membrane anchor. In the first catalytic step the carboxyl group of the substrate is converted to a kinetically activated carboxylate in N-carboxybiotin. After swing-over to the decarboxylase, an electrochemical Na(+) gradient is generated; the free energy of the decarboxylation is used to translocate 1-2 Na(+) from the inside to the outside, whereas the proton comes from the outside. At high [Na(+)], however, the decarboxylases appear to catalyse a mere Na(+)/Na(+) exchange. This finding has implications for the life of P. modestum in sea water, which relies on the synthesis of ATP via Delta(mu)Na(+) generated by decarboxylation. In many sequenced genomes from Bacteria and Archaea homologues of the carboxybiotin decarboxylase from A. fermentans with up to 80% sequence identity have been detected.     

Subunit composition of oxaloacetate decarboxylase and characterization of the alpha chain as carboxyltransferase

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits alpha, beta, gamma with Mr 65 000, 34 000 and 12 000, respectively. On dodecylsulfate/polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high-performance liquid chromatography in a dodecylsulfate-containing buffer. Biotin was localized exclusively in the alpha chain. Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the alpha chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin-Sepharose. No beta or gamma chain were detectable in this alpha chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated alpha chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [1-14C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of Na+ ions. The ease with which the alpha chain was released from the membrane characterize this subunit as a peripheral membrane protein. The beta and gamma chain, on the other hand, stick so firmly in the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins. Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the alpha chain, yielding a polypeptide of Mr 51 000 which was devoid of biotin. Degradation of the beta chain required prolonged incubation periods and was markedly influenced by Na+ ions which had a protective effect against proteolysis. A proton is required in the decarboxylation of oxaloacetate and CO2 arises as primary product. The other alternative, i.e. generation of HCO3- with H2O as substrate, has been excluded.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Site-directed sulfhydryl labeling of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae: helix VIII comprises a portion of the sodium ion channel

Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.     

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

The mechanism of oxaloacetate decarboxylase of Klebsiella aerogenes was investigated by enzyme kinetic methods. The activity of the decarboxylase was strictly dependent on the presence of Na+ or Li+ ions. For Li+ the Km was about 17 times higher and the Vmax about 4 times lower than for Na+. No activity was detectable at Na+ concentrations less than 5 microM. The curve for initial velocity versus Na+ concentration was hyperbolic. Initial velocity patterns with oxaloacetate or Na+ as the varied substrate at various fixed concentrations of the cosubstrate produced a pattern of parallel lines which is characteristic for a ping-pong mechanism. Product inhibition by pyruvate was competitive versus oxaloacetate and noncompetitive versus Na+. Oxalate, a dead-end inhibitor, was competitive versus oxaloacetate and uncompetitive versus Na+. The inhibition patterns are not consistent with a ping-pong mechanism comprising a single catalytic site but are analogous to kinetic patterns observed with the related biotin enzyme transcarboxylase, for which a catalytic mechanism at two different and independent sites has been demonstrated. The kinetic and other data support an oxaloacetate decarboxylase mechanism at two different sites of the enzyme with the intermediate formation of a carboxybiotin-enzyme complex. The first site is the carboxyltransferase which is localized on the alpha chain and the second site is the carboxybiotin-enzyme decarboxylase which is probably localized on the beta and/or gamma subunit. Binding studies with oxalate indicated that this is bound with high affinity to the alpha chain. The affinity was not affected by Na+ or by complex formation with the beta and gamma subunits. Oxalate protected the decarboxylase from heat inactivation but not from tryptic hydrolysis. The carboxybiotin-enzyme intermediate prepared from oxaloacetate decarboxylase with high specific activity was rapidly decarboxylated in the presence of Na+ ions alone. The effect of pyruvate on this reaction, noted previously, probably results from inhomogeneity of the enzyme preparation used which contained a considerable amount of free alpha subunits.     

The sodium ion translocating glutaconyl-CoA decarboxylase from Acidaminococcus fermentans: cloning and function of the genes forming a second operon

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans (clostridal cluster IX), a strict anaerobic inhabitant of animal intestines, uses the free energy of decarboxylation (delta G(o) approximately -30 kJ mol-1) in order to translocate Na+ from the inside through the cytoplasmic membrane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The largest subunit, alpha or GcdA (65 kDa), catalyses the transfer of CO2 from glutaconyl-CoA to biotin covalently attached to the gamma-subunit, GcdC. The beta-subunit, GcdB, is responsible for the decarboxylation of carboxybiotin, which drives the Na+ translocation (approximate K(m) for Na+ 1 mM), whereas the function of the smallest subunit, delta or GcdD, is unclear. The gene gcdA is part of the 'hydroxyglutarate operon', which does not contain genes coding for the other three subunits. This paper describes that the genes, gcdDCB, are transcribed in this order from a distinct operon. The delta-subunit (GcdD, 12 kDa), with one potential transmembrane helix, probably serves as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexible stretch of 50 amino acid residues (A26-A75), which consists of 34 alanines, 14 prolines, one valine and one lysine. The beta-subunit (GcdB, 39 kDa) comprising 11 putative transmembrane helices shares high amino acid sequence identities with corresponding deduced gene products from Veillonella parvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarchaeota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella typhimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobacteria). Directly upstream of the promoter region of the gcdDCB operon, the 3' end of gctM was detected. It encodes a protein fragment with 73% sequence identity to the C-terminus of the alpha-subunit of methylmalonyl-CoA decarboxylase from V. parvula (MmdA). Hence, it appears that A. fermentans should be able to synthesize this enzyme by expression of gctM together with gdcDCB, but methylmalonyl-CoA decarboxylase activity could not be detected in cell-free extracts. Earlier observations of a second, lower affinity binding site for Na+ of glutaconyl-CoA decarboxylase (apparent K(m) 30 mM) were confirmed by identification of the cysteine residue 243 of GcdB between the putative hellces VII and VIII, which could be specifically protected from alkylation by Na+. The alpha-subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.     

Entrance port for Na(+) and K(+) ions on Na(+),K(+)-ATPase in the cytoplasmic loop between trans-membrane segments M6 and M7 of the alpha subunit. Proximity Of the cytoplasmic segment of the beta subunit

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the alpha subunit of Na(+),K(+)-ATPase acts as an entrance port for Na(+) and K(+) ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na(+),K(+)-ATPase, and in parallel inactivates Rb(+) occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca(2+), Mg(2+), La(3+), p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo-2,4, 6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br(2)-TITU(3+)). 3) Ca(2+) ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha subunit. It appears that negatively charged residues in L6/7 recognize either Na(+) or K(+) ions or the competitive cation antagonists. Na(+) and K(+) ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the beta subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the beta subunit to 15-kDa at 20 degrees C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309-10316) markedly reduces affinity for Br(2)-TITU(3+) and for Na(+) ions, detected by Na(+) occlusion assays or electrogenic Na(+) binding, whereas Rb(+) occlusion is unchanged. 2) Na(+) ions specifically protect the 16-kDa fragment against this chymotryptic cleavage.     

The extracellular domain of the beta1 subunit is both necessary and sufficient for beta1-like modulation of sodium channel gating.

The type IIA voltage-gated sodium Na(+) channel from rat brain is composed of a large, pore-forming alpha subunit and the auxiliary subunits beta1 and beta2. When expressed in Xenopus oocytes, the beta1 subunit modulates the gating properties of the type IIA alpha subunit, resulting in acceleration of both inactivation and recovery from inactivation and in a negative shift in the voltage dependence of fast inactivation. The beta1 subunit is composed of an extracellular domain with a single immunoglobulin-like fold, a single transmembrane segment, and a small intracellular domain. A series of chimeras with exchanges of domains between the Na(+) channel beta1 and beta2 subunits and between beta1 and the structurally related protein myelin P0 were constructed and analyzed by two-microelectrode voltage clamp in Xenopus oocytes. Only chimeras containing the beta1 extracellular domain were capable of beta1-like modulation of Na(+) channel gating. Neither the transmembrane segment nor the intracellular domain was required for modulation, although mutation of Glu(158) within the transmembrane domain altered the voltage dependence of steady-state inactivation. A truncated beta1 subunit was engineered in which the beta1 extracellular domain was fused to a recognition sequence for attachment of a glycosylphosphatidylinositol membrane anchor. The beta1(ec)-glycosylphosphatidylinositol protein fully reproduced modulation of Na(+) channel inactivation and recovery from inactivation by wild-type beta1. Our findings demonstrate that extracellular domain of the beta1 subunit is both necessary and sufficient for the modulation of Na(+) channel gating.     

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.     

Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.     

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.