Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate

K _m for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as V _max was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PAT1, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (K _s) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 ± 3.20 and 3.73 ± 1.99 mM, respectively. It was shown that L-β-(N-benzylamino)alanine was a competitive inhibitor with respect to L-phenylalanine for PAT1 (K _i = 0.32 ± 0.07 mM, K _m = 0.45 ± 0.1 mM, V _max = 11. 6 ± 0.4 U/mg) at 25 mM concentration of 2-ketoglutarate in the reaction medium. L-β-(N-methylamino)alanine is a noncompetitive inhibitor with respect to L-phenylalanine for PAT3 (K _I = 138.4 ± 95.4 mM, K _m = 13.7 ±3.9 mM, V _max = 18.6 ± 4.1 U/mg) at 2 mM concentration of 2-ketoglutarate in the reaction medium. L-stereo isomers of nonprotein analogues of aromatic amino acids were studied as substrates for PAT1, PAT2, and PAT3. L-β-(2-Br-phenyl)alanine, L-β-(4-Br-phenyl)alanine, L-β-(2-F-phenyl)alanine, and L-(2-F)tryptophan were good substrates for all three aminotransferases; L-α-methyl-β-(2-Br-phenyl)alanine and L-O-benzyltyrosine were substrates only for PAT3; L-β-(4-F-phenyl)alanine was a substrate for PAT1 and PAT3. Thus, these analogues of aromatic amino acids can be stereoselectively synthesized using the studied aminotransferases in the presence of the corresponding keto acids.     

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0°C

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0°C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux K_m = 3.4 mM, V_max = 5.5 mM/min; infinite-trans efflux K_m = 8.7 mM, V_max = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux K_m = 2.7 mM, V_max = 2.4 mM/min; infinite-trans efflux K_m = 19 mM, V_max = 23 mM/min. The K_m values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474–484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux K_m values but somewhat lower V_max values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235–249). The results presented here support the adequacy of the carrier model to account for the kinetics.     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Hypoxanthine and thymidine compete for transport in Chinese hamster fibroblasts

The transport of thymidine and hypoxanthine was investigated in mutant Chinese hamster lung fibroblasts deficient in both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase. Kinetic data from rapid uptake experiments (0.5–4.5 s) indicate that thymidine is transported by a monophasic saturable system (K_m = 0.29 mM, V = 6.7 nmol/min · mg) which is competitively inhibited by hypoxanthine (K_i = 3.3 mM). The cells displayed a single transport system for hypoxanthine (K_m = 2.0 mM, V = 8.9 nmol/min · mg) that is inhibited competitively by thymidine (K_i = 0.43 mM). Both hypoxanthine and thymidine entry were noncompetively inhibited by nitrobenzylthioinosine, but thymidine transport was more sensitive. A kinetic model in which hypoxanthine and thymidine share a common transporter can account for the competitive inhibition and the observation that the inhibition constants are similar to the Michaelis constants.     

A novel low molecular weight alanine aminotransferase from fasted rat liver

Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with K_m values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free-SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a K_m of 0.057 mM.     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

The metabolism of nitrosopyrrolidine by hepatocytes from Fischer rats

Nitrosopyrrolidine (NO-PYR), an hepatocellular carcinogen, is rapidly metabolized to CO₂ by hepatocytes freshly isolated from the livers of male Fischer rats. Using CO₂ evolution as a measure of NO-PYR metabolism, we observed two kinetic constants; a high affinity component (K_m = 0.11 mM), and a lower affinity component (K_m = 3.2 mM). The high affinity component has similar kinetic constants to those observed for in vitro reactions with microsomes plus cytosol (K_m = 0.36 mM). Therefore, it is probable that the microsomal reaction is the limiting factor in the metabolism of NO-PYR in hepatocytes. NO-PYR may be metabolized to CO₂ through normal anaplerotic sequences. Some metabolites of NO-PYR which have been tentatively identified are γ-hydroxybutyrate, succinic semialdehyde, 3,4-dihydroxybutyric acid lactone, lactate, acetate, pyruvate, glyoxylate, γ-aminobutyrate and alamine. 2-Hydroxytetrahydrofuran (2-hydroxy-THF), a product of α-hydroxylation was detected at low levels in only one of four reactions. 3-Hydroxy-NO-PYR is present but represents only a small percentage of the total metabolism and is probably of little significance in the overall catabolism of NO-PYR in hepatocytes.     

Purification and characterization of methylglyoxal reductase from Hansenula mrakii

Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (K_m = 1.92 mM) and NADPH (K_m = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl₂. NADP also inhibited the activity of the enzyme, and the K_i value was calculated to be 0.25 mM.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

ATP-dependent K+ uptake by a photosynthetic purple sulfur bacterium

Chromatium vinosum contains an ATP-dependent K⁺ uptake system which is light independent and sensitive to DCCD. Kinetic measurements show K_m = 0.27 mM for K⁺ and K_m = 1.3 mM for Tl⁺, an alternate substrate. The internal K⁺ content of the cell increases with increasing medium osmolality and is unaffected by changes in external K⁺ concentration.     

In vitro prothrombin synthesis from a purified precursor protein III. Preparation of an acid-soluble substrate for vitamin K-dependent carboxylase by limited proteolysis of bovine descarboxyprothrombin

Bovine descarboxyprothrombin and descarboxyfragment-1 can be used as substrates for rat and bovine vitamin K-dependent carboxylase. In both enzyme systems, however, these substrates have a high K_m (0.3–0.4 mM). A better substrate (K_m = 0.001–0.003 mM) was prepared from bovine descarboxyprothrombin by limited proteolysis with subtilisin Carlsberg. This substrate is called Fragment-Su and is composed of the amino acids 13–29 of descarboxyprothrombin.     

The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0

The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 μM to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K_m values for the cofactor of 16 μM (low K_m) and 2.3 mM (high K_m). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V_max values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K_m form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K_m form with very little change in the overall V_max. A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Na+-Coupled Alanine Transport in LLC-PK1 Cells: The Relationship Between the K m for Na+ at Low [Alanine] and Potential Dependence for the System

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (K _m and V _max ) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na⁺/alanine cotransporter in LLC-PK₁ cells (1:1 Na⁺ to substrate coupling, and an ordered binding sequence), the algebraic expressions for K _m for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of K _m of the first-bound substrate (Na⁺) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the K ^Na _m includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na⁺, and (ii) conformational `translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent K ^Na _m for the LLC-PK₁ alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for K ^Na _m at 5 μm alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system. Received: 10 April 1998/Revised: 6 July 1998     

Separation and characterization of four hexose kinases from developing maize kernels

Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K_m values [K_m(glucose) = 117 micromolar, K_m(ATP) = 66 micromolar] whereas the other form had much higher K_m values [K_m(glucose) = 750 micromolar, K_m(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K_m(fructose) was about 130 micromolar and the K_m(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K_i(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ.     

Characterization and Regulation of Sulfur Reductase Activity in Thermotoga neapolitana

The growth of the hyperthermophilic, anaerobic bacterium Thermotoga neapolitana is stimulated by elemental sulfur by an unknown mechanism. We detected hydrogen-dependent sulfur reductase (sulfhydrogenase) and polysulfide dehydrogenase activities in cell extracts of this organism, demonstrating that it has at least two pathways for sulfidogenesis. Hydrogen-dependent sulfur reductase and hydrogenase activities are catalyzed by the purified hydrogenase of Thermotoga maritima, and this enzyme was called the sulfhydrogenase (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). Cells grown without elemental sulfur or cystine had 1.3 to 3.3 times higher sulfhydrogenase activities than those grown with either of these sources of sulfane sulfur. Hydrogenase activity was 2 to 5 times higher. Polysulfide dehydrogenase was up to 48-fold more active in cell extracts than the sulfhydrogenase. The activity of polysulfide dehydrogenase was approximately twofold higher when cells were grown in the presence of elemental sulfur. Its activity was oxygen labile in crude extracts, and it appears to be a cytoplasmic enzyme. Polysulfide was preferred over elemental sulfur as an electron acceptor (K_m = 0.15 mM) and was more active with NADH (K_m = 0.03 mM) than NADPH (K_m = 0.41 mM). Growth in the presence of elemental sulfur appeared to slightly increase the activity of polysulfide dehydrogenase and slightly decrease both activities of sulfhydrogenase (hydrogenase and polysulfide reductase), while growth without elemental sulfur had the opposite effects. The greater activity of polysulfide dehydrogenase and its apparent regulation indicate that it is the more physiologically important means of polysulfide reduction.     

Catalytic properties of aminoacylase of strain <Emphasis Type="Italic">Rhodococcus armeniensis</Emphasis> AM6.1

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K_m) and maximum reaction velocities (V_max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K_s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K_s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K_i = 104.7 ± 21.7 mM, K_m = 2.5 ± 0.5 mM, V_max = 25.1 ± 1.5 U/mg) was observed.     

Sodium-dependent activation of intestinal brush-border sucrase: Correlation with activation by deprotonation from pH 5 to 7

The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK₁ of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK₂ of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V_m and K_m. The V_m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK_1y, about 5.6) was little affected by sodium. On the contrary, the K_m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK_1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541).     

Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO₂, and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K_m values for both NAD⁺ (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from ~7.5 to ~6.4 when the MgCl₂ and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl₂ concentration. MgCl₂ was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate.