On the mechanism of sodium ion translocation by methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7-8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating delta psi of 60 mV, delta pNa+ of 50 mV, and delta mu Na+ of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

A biotin-dependent sodium pump: glutaconyl-CoA decarboxylase from Acidaminococcus fermentans

The decarboxylation of glutaconyl-CoA to crotonyl-CoA in the anaerobic bacterium Acidaminococcus fermentans is catalysed by a membrane-bound, biotin-dependent enzyme which requires Na+ for activity. Inverted vesicles from A. fermentans accumulated Na+ only if glutaconyl-CoA was decarboxylated. The Na+ uptake was inhibited by avidin but not by the avidin biotin complex. Detergents and ionophores such as monensin also prevented the Na+ transport. The results indicate that the enzyme is able to convert the free energy of decarboxylation (delta Go' approximately equal to -30 kJ/mol) into a Na+ gradient.     

Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X-100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate-binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X-100 and purified by affinity chromatography on monomeric avidin-Sepharose. The enzyme is composed of three types of polypeptides: the group of alpha chains (Mr 120000-140000) containing the biotin, the beta chain (60000) and an apparently hydrophobic gamma chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The beta and gamma chains but not the alpha chain could be labelled by N-ethyl-[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000-20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl-CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.     

Stereochemistry of the methylmalonyl-CoA decarboxylation reaction

The steric course of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA, catalyzed by the biotin-dependent sodium pump methylmalonyl-CoA decarboxylase of Veillonella alcalescens was determined. The decarboxylation of (S)-methylmalonyl-CoA in 3H2O yielded (R)-[2-3H]propionyl-CoA; and the decarboxylation of (S)-[2-3H]methylmalonyl-CoA in H2O produced (S)-[2-3H]propionyl-CoA. The results demonstrate retention of configuration during the decarboxylation reaction. The substrate stereochemistry of methylmalonyl-CoA decarboxylase is thus the same as that of all other biotin-containing enzymes investigated.     

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.     

The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans was inactivated by incubation with n-alkanols at 37 degrees C. The concentration of the alcohol required for complete inactivation decreased with increasing chain length; e.g. 2 M ethanol was as potent as 2 mM hexanol or 0.5 mM decanol. The data indicate a binding of the alcohol to the enzyme with an energy of about 4 kJ/methylene group. Sodium ions prevented the inactivation (50% at 30 mM NaCl). K+, NH4+, Cs+ and Mg2+ had no influence, whereas Li+ was ten times less effective than Na+. The enzyme was cleaved during the inactivation into a soluble part, consisting of the alpha (Mr 120,000) and beta polypeptide chains (60,000), whereas the hydrophobic gamma chain (30,000) precipitated. The soluble part catalysed the sodium-ion-independent but avidin-sensitive glutaconyl-CoA/crotonyl-CoA exchange as measured with the substrates [3-3H]crotonyl-CoA and unlabelled glutaconate and with glutaconate CoA-transferase as auxiliary enzyme. In the presence of free biotin or its methyl ester the soluble part catalysed the formation of crotonyl-CoA from glutaconyl-CoA (apparent Km for biotin 40 mM, Vmax 1% of the native decarboxylation reaction). This apparent reactivation was most likely caused by the carboxylation of free biotin. Based on these and other observations the following functions may be assigned to the different polypeptide chains of glutaconyl-CoA decarboxylase: biotin carrier (alpha), carboxytransferase (beta) and carboxylase, the actual sodium pump (gamma).     

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.     

Malonyl-CoA decarboxylase from Streptomyces erythreus: purification, properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an alpha 2 subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a Km of 143 microM and V of 250 nmol min-1 mg-1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the (R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the (S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.     

Properties of oxaloacetate decarboxylase from Veillonella parvula.

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.     

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.     

Malonyl-CoA decarboxylase from Mycobacterium tuberculosis and Pseudomonas fluorescens.

Malonyl-CoA decarboxylase was partially purified (nearly 1000-fold) from Mycobacterium tuberculosis H37Ra by ammonium sulfate precipitation, gel filtration with Sepharose 6B, and chromatography on DEAE Sephacel, carboxymethyl-Sephadex, and NADP-agarose. Polyacrylamide gel electrophoresis showed a major band (60–70%), which contained the enzymatic activity, and a minor band which had no decarboxylase activity. The molecular weight of the enzyme was 44,000, and the PI and pH optimum were 6.7 and 5.5, respectively. The enzyme showed a typical Michaelis-Menten substrate saturation, with an apparent K_m and V of 0.2 mm and 3.85 μmol/min/mg, respectively. It catalyzed decarboxylation of methylmalonyl-CoA only at 5% of the rate observed with malonyl-CoA, whereas malonic acid and succinyl-CoA were not decarboxylated. Antibodies prepared against malonyl-CoA decarboxylase from the uropygial glands of goose and rat liver mitochondria did not inhibit the bacterial enzyme. Avidin did not inhibit the enzyme suggesting that biotin was not involved in the reaction. Thiol-directed reagents inhibited the enzyme as did CoA, acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Malonyl-CoA decarboxylase was also partially purified from malonate-grown Pseudomonas fluorescens. The molecular weight of this enzyme was 56,000 and the pH optimum and apparent K_m were 5.5 and 1 mm, respectively. Unlike the mycobacterial enzyme, this enzyme was insensitive to p-hydroxymercuribenzoate, acetyl-CoA, and propionyl-CoA, and it was less sensitive to inhibition by succinyl-CoA and CoA than the mycobacterial enzyme. The size and properties of the two bacterial enzymes suggest that these are quite unlike the mammalian and avian enzymes and that they constitute a different class of malonyl-CoA decarboxylases.     

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.     

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.     

Sodium-calcium exchange in membrane vesicles from Artemia

Membrane vesicles were prepared from Artemia nauplii (San Francisco Bay variety) 45 h after hydration of the dry cysts. Na+-loaded vesicles accumulated up to 10 nmol Ca2+/mg protein when diluted 50-fold into 160 mM KCl containing 15 microM CaCl2. Practically no accumulation of Ca2+ was observed if the vesicles were diluted into 160 mM NaCl instead of KCl, or if they were treated with monensin, a Na+ ionophore, for 30 s prior to addition of CaCl2 to the KCl medium. These observations indicate that the Artemia vesicles exhibit Na-Ca exchange activity. The velocity of Ca2+ accumulation by the vesicles in KCl was stimulated 2.6-fold by the K+ ionophore valinomycin, suggesting that the exchange system is electrogenic, with a stoichiometry greater than 2Na+ per Ca2+. Km,Ca and Vmax values were 15 microM and 7.5 nmol/mg protein.s, respectively. Exchange activity in the Artemia vesicles was inhibited by benzamil (IC50 approximately equal to 100 microM) and by quinacrine (IC50 approximately equal to 250 microM), agents that also inhibit exchange activity in cardiac sarcolemmal vesicles. Unlike cardiac vesicles, however, exchange activity in Artemia was not stimulated by limited proteolysis, redox reagents, or intravesicular Ca2+. This indicates that the two exchange systems are regulated by different mechanisms. Vesicles were prepared from Artemia at various times after hydration of the dry cysts and examined for exchange activity. Activity was first observed at approximately 10 h after hydration and increased to a maximal value by 30-40 h; hatching of the free swimming nauplii occurred at 18-24 h. The results suggest that hatching Artemia nauplii might be a particularly rich source of mRNA coding for the Na+-Ca2+ exchange carrier.     

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.     

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.     

Purification and characterization of a new NAD(+)-dependent enzyme, L-tartrate decarboxylase, from Pseudomonas sp. group Ve-2.

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.     

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane.

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.     

Substrate stereochemistry of the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans

The steric course of the decarboxylation of glutaconyl-CoA to crotonyl-CoA, catalysed by the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans, was elucidated using the sequence: chiral acetate----citrate----glutamate----glutaconyl-CoA----crotonyl-CoA ----chiral acetate. Since glutaconyl-CoA or glutaconate labeled at C-4 was subjected to rapid chemical or enzymatic exchanges, glutamate was fermented to acetate by growing cells of A. fermentans. The analysis of the final chiral acetates gave following deviations from 50% in the fumarase exchange: + 13.8% starting with (R)-acetate and - 13.9% starting with (S)-acetate. The results demonstrated a retention of configuration during the decarboxylation. Thus glutaconyl-CoA decarboxylase adds to the list of biotin enzymes in which exclusive retention of configuration was observed. Glutaconate CoA-transferase from A. fermentans catalysed a 3H exchange of [2,4,4-3H]glutaconate with water when acetyl-CoA was present. At low concentration of acetyl-CoA (20 microM) the exchange ceased after exactly one atom 3H was released into the water, at high concentrations (1 mM) the exchange proceeded further. The apparent Km of acetyl-CoA in the exchange (1.1 microM) was 150 times smaller than that of the complete CoA transfer. It was concluded that either a mixed anhydride, between a carboxyl group of the enzyme and [2,4,4-3H]glutaconate, or enzyme-bound glutaconyl-CoA was the exchanging species.