Carbonic acid from decarboxylation by "malic" enzyme in lactic acid bacteria

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of l-malic acid by "malic" enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and l-malic acid, at pH 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from "malic" enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

Selective induction of bilirubin UDP-glucuronosyl-transferase by perfluorodecanoic acid

Differential effects of perfluorodecanoic acid (PFDA) on rat liver UDP-glucuronosyltransferase isoenzymes have been observed after a single i.p. administration of the compound to young male Sprague-Dawley rats. (1) Bilirubin glucuronidation was induced 2-fold. The induced state was stable for at least 3 weeks. (2) Glucuronidation of 1-naphthol, morphine and testosterone was decreased to half of the control values. These decreases were maximal after 12 days but all three activities returned to normal levels after 3 weeks. (3) Immunoblotting experiments indicated that the differential effects of PFDA on UDP-glucuronosyltransferase activities were due to modulation of enzyme protein concentrations rather than activation/inactivation mechanisms. With respect to its influence on UDP-glucuronosyltransferase isoenzymes, PFDA may be classified as a clofibrate-type inducer. The persistence of the induction after a single application however is unique among peroxisome proliferators and therefore PFDA may be a useful, elective inducer of bilirubin glucuronidation.     

A dual mechanism regulates the insulin stimulation of hepatic malic enzyme

The activity of malic enzyme, an important hepatic lipogenic enzyme, is stimulated in diabetic rats by insulin administration. This process was shown to involve increases in both enzyme quantity and the specific activity (units activity/nmol enzyme) of the enzyme. Therefore, the coupling of these two regulatory mechanisms was responsible for the insulin-mediated increase in malic enzyme activity.     

Dietary induction of hepatic malic enzyme activity: differentiation of the induction process

The activity of rat liver malic enzyme shows a marked increase when the animals are maintained on a restricted protein diet. Of the NADP-linked dehydrogenases tested (malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase), the response is confined only to malic enzyme. Dietary sucrose is not required for the increase in activity, but elevated dietary levels of this disaccharide increase hepatic malic enzyme regardless of dietary protein. Glucose-6-phosphate dehydrogenase activity is increased by dietary sucrose provided adequate dietary protein is supplied. The specificity of the response to lowered dietary protein shown by malic enzyme suggests that the control of the hepatic enzyme is mediated by processes different from those controlling the activity of glucose-6-phosphate dehydrogenase.     

Pigeon liver malic enzyme

Summary Malic enzyme of pigeon liver is a tetrameric molecule with identical, or nearly-identical subunits. It catalyzes, in addition to oxidative decarboxylation of L-malate, the following metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on -ketocarboxylic acids; a NADP⁺-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water. The kinetic mechanism of oxidative decarboxylase is sequential and ordered, with NADP⁺ adding first to the metal enzyme, followed by L-malate, and by the release of products CO₂, pyruvate, and NADPH. NADPH release, or a conformation change preceeding it, is rate-limiting in the overall reaction.Chemical modification studies indicate the presence of histidyl and lysyl residues at the nucleotide site, and tyrosyl residues at the carboxylic acid site. The involvement of protonated histidine(s) in NADPH binding is implicated by results of direct titration experiments, which also suggest a role of this residue as a proton sink in the catalytic reaction.A cysteinyl SH group is located near (but not at) each of the substrate-sites on the enzyme tetramer. Reaction of these groups with SH reagents causes selective loss of activities involving decarboxylation (i.e., oxidative decarboxylase, reductive carboxylase, and oxalacetate decarboxylase), owing to blockage of the reversible carbon-carbon cleavage step by the bulky substituent. All-of-the-sites reactivity is observed for non-specific thiol reagents such as 5,5 dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetate, and iodoacetamide. While bromopyruvate, which is reduced by the enzyme to L-bromolactate under catalytic conditions, alkylates these groups in an active-site directed manner with half-of-the-sites stoichiometry. The remaining two SH groups are reactive toward non-specific reagents, but at rates 2.4 - 3.6 fold lower than do the same groups on the unalkylated enzyme. This behavior is interpreted in terms of the ligand-induced negative cooperativity concept of Koshland, et al. (Biochemistry 5: 365–385, 1966): Reaction of bromopyruvate induces a conformation change on the alkylated subunit which is transmitted to the unoccupied subunit neighbor, turning off its catalytic site for reaction with L-malate, as well as converting the initial fast SH groups into slow, or unreactive SH groups.In equilibrium binding experiments, all-of-the-sites reactivity is seen with nucleotide cofactors NADP⁺ and NADPH. Binding of Mn²⁺, or L-malate in the presence of Mn²⁺ and NADPH is biphasic, showing two tight sites with dissociation constants in the micromolar range, and two weak sites with 10–100 fold lower affinities. The presence of tight and weak L-malate sites is confirmed by fluorescence titration experiments which also yields similar affinities for the substrate molecule. In kinetic studies, two types of non-equivalent, and functionally distinct sites are detected. At saturating NADP⁺, and Mn²⁺ and L-malate levels corresponding to binding at tight sites, typical Michaelian behavior is observed. The reaction is inhibited uncompentitively by L-malate at higher concentrations corresponding to occupancy at all of the L-malate sites. Occupancy of Mn²⁺ at weak metal sites as well has no effect at low L-malate, but prevents substrate inhibition at high L-malate.A tentative half-of-the-sites model consistent with results of chemical modification, binding, and kinetic experiments is proposed for this enzyme. This model implicates involvement of subunit cooperativity in the catalytic process. Malic enzyme is depicted as a tetramer composed of inititally identical subunits, each containing an active-site capable of binding all reactants. Mn²⁺ and L-malate bind anticooperatively to the tight and weak sites, in contrast to NADP⁺ which binds equivalently to all sites. On the fully active enzymes, only half (or the tight) of the subunits are simultaneously undergoing catalysis. Binding of L-malate (but not Mn²⁺) at the adjacent weak subunits causes a slow isomerization of the enzyme, and inhibition of NADPH dissociation from the catalytic subunits. Binding of Mn²⁺ at the same sites prevents this change and thereby relieving substrate inhibition. This model is further supported by results of active-site titration experiments, such as the half-size burst of enzyme-bound NADPH in the transient state, and half-of-the-sites reactivity of oxalate, an analog for the transition state intermediate of the reaction.Abbreviations DTNB 5,5 dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - BP bromopyruvate - DTT dithiothreitol     

Insulin stimulation of hepatic malic enzyme activity in normal and diabetic rats controlled by different regulatory processes

A comparison of the processes controlling the increase in hepatic malic enzyme activity in insulin-treated normal and diabetic rats indicated the existence of two distinct regulatory mechanisms. Livers were removed at 12, 36, and 60 h after insulin treatment of normal and alloxan-diabetic rats, and the activity, quantity, and specific activity (units/nmol), of malic enzyme was determined. In normal rats, a significant increase in activity occurred 12 h after insulin, whereas 36 h of insulin treatment was required for diabetic rats to show an increase in enzyme activity. This suggested that the return of malic enzyme activity from the depleted levels measured in diabetic rats probably involved a different sequence of events. A malic enzyme specific radioimmunoassay confirmed this. The increase in activity in insulin-treated normal rats was due to an increase in the quantity of malic enzyme. In insulin-treated diabetic rats, the increase in activity resulted from increases in both enzyme quantity and the specific activity of the enzyme, which returned to levels observed in normal rats.     

Purification of pigeon liver malic enzyme by affinity chromatography

A simple and rapid method for the purification of malic enzyme (EC 1.1.1.40) from pigeon liver is described. Malic enzyme in the crude tissue extract was partially purified by heat treatment, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Final purification was achieved by affinity chromatography on immobilized N⁶-(6-aminohexyl)-adenosine 2′,5′-bisphosphate. Apparently homogeneous enzyme was obtained in 2 days with 54% yield.     

Influence of pH,malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae

Malic acid consumption by Saccharomyces cerevisiae was studied in a synthetic medium. The extent of malic acid degradation is affected by its initial concentration, the extent and the rate of deacidification increased with initial malate concentration up to 10 g/l. For malic acid consumption, an optimal pH range of 3–3.5 was found, confirming that non-dissociated organic acids enter S. cerevisiae cells by simple diffusion. A full factorial design has been employed to describe a statistical model of the effect of sugar and malic acid on the quantity of malate degraded (g/l) by a given amount of biomass (g/l). The results indicated that the initial malic acid concentration is very important for the ratio of malate consumption to quantity of biomass. The yeast was found to be most efficient at higher levels of malate.