Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.     

Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide

Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.     

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and characterization of hydroxypyruvate reductase from the facultative methylotroph Methylobacterium extorquens AM1.

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Characterization of two carnosine-degrading enzymes from rat brain. Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis

Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.     

Mitochondrial and cytosolic hexokinase from rat brain: one and the same enzyme?

Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.     

Purification and partial characterization of dimeric dihydrodiol dehydrogenase from monkey kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of several monkey tissues, among which kidney exhibited the highest activity and contained a high-molecular weight (Mr approximately 65,000) enzyme species. The enzyme species was purified to apparent homogeneity and showed a subunit molecular weight of 39,000. The enzyme oxidized benzene dihydrodiol (Km = 0.9 mM) at a pH optimum of 9.8, and highly reduced vicinal diketones such as camphorquinone (Km = 0.1 mM) and diacetyl (Km = 0.8 mM) around pH 7.5, but alicyclic alcohols, hydroxysteroids and ketosteroids were inactive substrates for this enzyme. Quercitrin, SH-reagents, stilbestrol were inhibitory to the enzyme activity, but other synthetic estrogens, anti-inflammatory agents and 3-ketosteroids were not.     

Histidine ammonia-lyase from rat liver. Purification, properties, and inhibition by substrate analogues.

Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.     

The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes

The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle.     

Monkey liver microsomal glucose-6-phosphatase]

Kinetic studies indicate that glucose-6-phosphatase is a multifunctional enzyme. a) Phosphohydrolase activities. The mannose-6-phosphatase activity is low (Km = 8 mM, VM = 90 nmoles. min-1mg-1). The enzyme shows a strong affinity for glucose-6-phosphate (Km = 2.5 mM, VM = 220 nmoles.min-1mg-1). beta-glycerophosphate (K1 = 30 mM), D-glucose (Ki = 120 mM) are mixed type inhibitors; pyrophosphate (Ki = 2 mM) is a non competitive one. b) Phosphotransferase activities. Di and triphosphate adenylic nucleosides or phosphoenol pyruvate are not substrates. Carbamylphosphate serves as a phosphoryl donor with D-glucose as acceptor. The phosphate transfer is consisstent with a random mechanism in which the binding of one substrate increases the enzymes affinity for the second substrate. Apparent Km values for carbamyl-phosphate range from 5.2 mM (D-glucose concentration leads to infinity) to 8 mM (D-glucose concentration leads to 0). The corresponding apparent Km values for D-glucose are 59 mM (carbamyl-phosphate concentration leads to infinity) to 119 mM (carbamyl-phosphate concentration leads to 0). Maximal reaction velocity with infinite levels of both substrates is 270 nmoles.min-1.mg-1. Pyrophosphate is a poor phosphoryl donnor (Km = 55 mM with D-glucose concentration 250 mM). In addition we do not find any latency; detergents, namely sodium deoxycholate, Triton X 100 do not affect or inhibit glucose-6-phosphatase activity.     

Lysosomal thyroid hormone 5''-deiodinase

The transglutaminase-mediated insertion of putrescine into casein was inhibited competitively by alpha-difluoromethylornithine (alpha-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Preincubation of the amine acceptor (casein) or the enzyme itself with the inhibitor did not affect enzyme activity. Alpha-DFMO is a poorer substrate for transglutaminase (Km = 2.10 mM) than putrescine (Km = 0.17 mM). The inhibitory effect was also found with fibronectin as amine acceptor.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

Activity of membranous dopamine beta-monooxygenase within chromaffin granule ghosts. Interaction with ascorbate

The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.     

The specificity of oxidase and kinase preparations from Pseudomonas fluorescens towards deoxyfluoromonosaccharides.

With partially purified enzyme preparations from cell-free extracts of Pseudomonas fluorescens, 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid are substrates for glucose oxidase (EC 1.1.3.4.) and gluconate dehydrogenase (EC 1.1.99.3), with K-m values 18.2 mM and 11.8 mM, respectively. The same enzymes that oxidize glucose and gluconic acid probably oxidize 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid. The latter fluorinated carbohydrates and the presumed formation of 3-deoxy-3-fluoro-2-keto-D-gluconic acid, which has been isolated as a calcium salt and characterizied, are not substrates for gluconokinase (EC 2.7.1.12). Both 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid act as competitive inhibitors of this enzyme preparation for gluconate, with K-i values 47.5 mM and 14.8 mM, respectively.     

Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.