Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67? omitted?000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21? omitted?000 and 13? omitted?000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.     

Purification and Characterization of Phosphoribosylpyrophosphate Synthetase from Rubber Tree Latex

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate, and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribulose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.     

Alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium. Selective activation by manganese and effect of other divalent cations.

1. Alkaline p-nitrophenylphosphate phosphatase (pNPPase) activity of Halobacterium halobium is selectively stabilized and stimulated by Mn2+ ions. 2. Mn2+ binding to native pNPPase is characterized by a dissociation constant of 0.35 mM at pH 8.5, 37 degrees C, with a Hill coefficient of 0.988. 3. Mn2+ behaves as a mixed type nonessential activator, increasing the Vmax value (beta = 6.09, pH 8.5) and decreasing the Km value for pNPP (alpha = 0.56, pH 8.5). The Ki value for inorganic phosphate (a competitive inhibitor) was also decreased in the presence of Mn2+. 4. Activation of native pNPPase by preincubation with Mn2+ is a slow temperature-dependent process, which can be described by an exponential relationship vs time. However, a weak but immediate activation was also detected. 5. Zn2+, Cu2+ and Ni2+ were found to inhibit both native and Mn(2+)-stimulated pNPPase, whereas Co2+ and Cd2+ inhibited the Mn(2+)-stimulated pNPPase but had no effect on the native enzyme form.     

The crystal structure of TrpD, a metabolic enzyme essential for lung colonization by Mycobacterium tuberculosis, in complex with its substrate phosphoribosylpyrophosphate

Mycobacterium tuberculosis, the cause of tuberculosis, presents a major threat to human health worldwide. Biosynthetic enzymes that are essential for the survival of the bacterium, especially in activated macrophages, are important potential drug targets. Although the tryptophan biosynthesis pathway is thought to be non-essential for many pathogens, this appears not to be the case for M.tuberculosis, where a trpD gene knockout fails to cause disease in mice. We therefore chose the product of the trpD gene, anthranilate phosphoribosyltransferase, which catalyses the second step in tryptophan biosynthesis, for structural analysis. The structure of TrpD from M.tuberculosis was solved by X-ray crystallography, at 1.9 A resolution for the native enzyme (R = 0.191, Rfree = 0.230) and at 2.3 A resolution for the complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg2+ (R = 0.194, Rfree = 0.255). The enzyme is folded into two domains, separated by a hinge region. PRPP binds in the C-terminal domain, together with a pair of Mg ions. In the substrate complex, two flexible loops change conformation compared with the apo protein, to close over the PRPP and to complete an extensive network of hydrogen-bonded interactions. A nearby pocket, adjacent to the hinge region, is postulated by in silico docking as the binding site for anthranilate. A bound molecule of benzamidine, which was essential for crystallization and is also found in the hinge region, appears to reduce flexibility between the two domains.     

Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli

The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 °C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 °C. The enzyme was stable at 4 °C for 25 days. The apparent K(m) values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD(+) . The k(cat) /K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD(+) . Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD(+) . EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn(2+) , Co(2+) , Zn(2+) , Ni(2+) and Cu(2+) . However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn(2+) , Ni(2+) and Cu(2+) . The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Biosynthesis of phosphatidylethanolamine by enzyme preparations from plant tissues

The enzymic utilization of cytidine diphosphoethanolamine in the synthesis of phosphatidylethanolamine is localized in the microsomal fraction of spinach (Spinacia oleracea) leaves. The metal ion requirement can be satisfied by Mn(2+) (saturation approximately 0.6 mm) or Mg(2+) (saturation approximately 25 mm). The enzyme has a pH optimum of 8.0 in the presence of Mn(2+) and 7.5 in the presence of Mg(2+). A Michaelis constant of 20 mum was determined for cytidinediphos-phoethanolamine. Enzyme activity was stimulated by thiol compounds and inhibited by thiol reagents. No inhibition was obtained with cytidine monophosphate and Tween 80.The in vitro biosynthesis of phosphatidylethanolamine was inhibited by cytidine diphosphocholine and the biosynthesis of phosphatidylcholine was inhibited by cytidine diphosphoethanolamine. Activities of the two synthetic systems were indistinguishable on the basis of susceptibility to lyophilization and inhibition by thiol reagents.     

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase

Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC(50) values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 μM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn(2+) at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn(A)(2+) and Mn(B)(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.     

A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

A specific sucrose phosphatase from plant tissues

1. A phosphatase that hydrolyses sucrose phosphate (phosphorylated at the 6-position of fructose) was isolated from sugar-cane stem and carrot roots. With partially purified preparations fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, glucose 1-phosphate and fructose 1,6-diphosphate are hydrolysed at between 0 and 2% of the rate for sucrose phosphate. 2. The activity of the enzyme is increased fourfold by the addition of Mg(2+) ions and inhibited by EDTA, fluoride, inorganic phosphate, pyrophosphate, Ca(2+) and Mn(2+) ions. Sucrose (50mm) reduces activity by 60%. 3. The enzyme exhibits maximum activity between pH6.4 and 6.7. The Michaelis constant for sucrose phosphate is between 0.13 and 0.17mm. 4. At least some of the specific phosphatase is associated with particles having the sedimentation properties of mitochondria. 5. A similar phosphatase appears to be present in several other plant species.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Slow binding of metal ions to pigeon liver malic enzyme: a general case

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-binding process, which resulted in a concave down tracing of the enzyme activity assay. The fast initial rates were independent of lutetium ion concentration, while the slow steady-state rates decreased with increasing Lu(3+) concentration. The observed rate constant for the transition from initial rate to steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu(3+) concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu(3+) to the R-form is weaker (K(d,Lu) = 14 microM) than that of Mn(2+) (K(m,Mn) = 1.89 microM); however, Lu(3+) has much tighter binding affinity with the T-form ( = 0.83 microM). Lu(3+) was shown to be a competitive inhibitor with respect to Mn(2+), which suggests that Lu(3+) and Mn(2+) are competing for the same metal binding site of the enzyme. These observations are in accordance with the available crystal structure information, which shows a distorted active site region of the Lu(3+)-containing enzyme. Other divalent cations, i.e., Fe(2+), Cu(2+), or Zn(2+), also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu(3+)-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. The above results indicated that replacement of the catalytically essential Mn(2+) by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed enzyme conformation, however, is unfavorable for catalysis. Both the chemical nature of the metal ion and its correct coordination in the active site are essential for catalysis.     

Effect of copper on acid phosphatase activity in yeast Yarrowia lipolytica

Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu2+ concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu2+, Co2+, Ni2+, Mn2+ and Mg2+ and inhibited by Ag+ and Cd2+. The most effective ion was Cu2+, especially for the enzyme from cultures in medium containing Cu2+, whereas APase activity in wall-bound fragments was only slightly activated by Cu2+. The content of cellular phosphate involving polyphosphate was decreased by adding Cu2+, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu2+ might depend on derepression of the gene encoding the APase isozyme.