Property of midgut α-amylase from Mythimna separata (Lepidoptera: Noctuidae) larvae and its responses to potential inhibitors in vitro.

Midgut α-amylase is an important digestive enzyme involved in larval energy metabolism and carbohydrate assimilation. In this article, the properties of midgut α-amylase from the Oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), larvae were characterized, and its in vitro responses to chemical inhibitors were also determined. The kinetic parameters Km and Vmax of midgut α-amylase were 0.064 M, 4.81 U mg pro(-1) in phosphate buffer, and 0.128 M, 1.96 U mg pro(-1) in barbiturate-acetate buffer; α-amylase activity linearly increased as starch concentration increased. α-Amylase activity was not influenced by amino acids such as Pro, Met, Try, His, Ala, and Phe but was strongly activated by antioxidants. Reduced glutathione, 1,4-dithiothreitol, ß-mercaptoethanol, and ascorbic acid improved the activity of α-amylase about 2.06, 3.46, 3.37, and 6.38 times, respectively, relative to the control. Ethylenediaminetetraacetic acid, sodium dodecyl sulfonate, and N-bromosuccinimide (NBS) strongly inhibited α-amylase. α-, ß-, and γ-cyclodextrin were not the preferred substrates for α-amylase. Kinetic analysis showed that IC50 value of NBS against α-amylase was 1.52 (±0.26) µM, and the mode of action of NBS with Ki as 2.53 (0.35) µM was a mixed-type inhibition that indicated a combination of partial competitive and pure noncompetitive inhibition. The midgut α-amylase of armyworm larvae may be a potential target for novel insecticide development and pest control.

Catalytic characteristics of plant-esterase from wheat flour.

A plant-esterase extracted from wheat flour and purified with a PEG1000/NaH(2)PO(4) aqueous two-phase system was characterized for its catalytic characteristics. The optimal condition for plant-esterase to catalyze 1-naphthyl acetate was at 30°C, pH 6.5. It kept stability at 20°C during 120 min and at pH 5.5 during 60 h. The effects of metal ions, chemical modification reagents and pesticides on plant-esterase activity were investigated. It was found that Ba(2+) and Pb(2+) at concentrations of 20 mM significantly inhibited the activity of plant-esterase while Mg(2+), Ca(2+) and Fe(2+) at the same concentration enhanced the enzyme activity. Chemical modification reagents significantly influenced the activity of plant-esterase. Particularly, PMSF (4.5 mM) and N-bromosuccinimide (11 mM) inhibited by 5.40-19.87% of the enzyme activity. It is implied that serine and tryptophan are related to the enzyme activity. Plant-esterase were displayed concentration-dependent inhibition by dichlorvos, carbofuran and carbendazim (IC50 = 0.31-63.12 ppm). All these results indicated that catalytic efficiency of plant-esterase strongly depends on reaction conditions, activity effectors and amino acid residues at the active site. It makes meaningful guidance on further design of sensing material in monitoring pesticides.

pH-dependent modulation of connexin-based gap junctional uncouplers.

Gap junction (GJ) channels formed from connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell­cell communication exhibiting high sensitivity to intracellular pH (pH(i)). We examined pH(i)-dependent modulation of junctional conductance (g(j)) of GJs formed of Cx26, mCx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols (LCCAs), arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH4Cl to pH ∼8 increased g(j) in cells expressing mCx30.2 and Cx45, yet did not affect g(j) of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 GJs. Unexpectedly, cells expressing Cx45, but not other Cxs, exhibited full coupling recovery after alkalization with NH4Cl under the continuous presence of LCCAs, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC50 for octanol was 0.1, 0.25 and 2.68 mm at pH(i) values of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide reduced the coupling-promoting effect of NH4Cl as well as the uncoupling effect of octanol. This suggests that LCCAs and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 GJ channel protein.

Purification of beta-galactosidase from Erythrina indica: involvement of tryptophan in active site.

beta-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected beta-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 degrees C, respectively. The enzyme showed a K(m) value of 2.6 mM and V(max) of 3.86 U/mg for p-nitrophenyl-beta-D-galactopyranoside as substrate and was inhibited by Zn(2+) and Hg(2+). The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by beta-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of beta-galactosidase. Circular dichroism studies revealed 37% alpha helix, 27% beta sheet and 38% random coil in the secondary structure of the purified enzyme.

Isolation and characterization of a novel acid proteinase, tropiase, from Candida tropicalis IFO 0589.

A novel acid proteinase (Tropiase) was isolated from Candida tropicalis IFO 0589 by DE52-cellulose, and DEAE-Cosmogel column chromatographies. The purified tropiase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme preparation had a molecular weight of 23,900, isoelectric point of pH 5.1, optimum pH range of 7 to 9 and possessed 208 amino acid residues. The enzyme hydrolyzed casein, fibrinogen, keratin and collagen. The purified tropiase demonstrated hemorrhagic and capillary permeability-increasing activities. Inhibition of tropiase occurred with leupeptin and N-bromosuccinimide, however, no inhibition was observed with alpha(2)-macroglobulin, soybean trypsin inhibitor, benzamidine-HCl or diisopropyl fluorophosphate.

Purification and Properties of Extracellular Lipases with Transesterification Activity and 1,3-Regioselectivity from Rhizomucor miehei and Rhizopus oryzae.

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at 40°C and pH 7.0 for the R. miehei lipase, and at 30°C and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the Vmax of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM Hg2+, Zn2+, or Mn2+, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly ß sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

Studies of the N-bromosuccinimide inactivation of the enzyme rhodanese.

The enzyme rhodanese (Thiosulfate: cyanide sulphurtransferase, EC 2.8.1.1) is rapidly inactivated by treatment with N-bromosuccinimide. Spectrophotometric titration and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that neither tryptophan oxidation nor polypeptide chain cleavage can account for the inactivation. Sulfhydryl group assays using the colormetric reagent 5,5'-dithiobis(2-nitrobenzoic acid) after destruction of excess N-bromosuccinmide, indicate that approximately 2 sulfhydryl groups per enzyme molecule are lost. Further, rhodanese inactivated by N-bromosuccinimide can be reactivated (approximately 95%) by incubation with the substrate thiosulfate. It is postulated that N-bromosuccinimide inactivates rhodanese by inducing the formation of a disulfide bond involving the active site sulfhydryl group of the enzyme and a second sulfhydryl group which can be brought close to the active site in the flexible native structure.

Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.

The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.

Characterisation of a xylanolytic amyloglucosidase of Termitomyces clypeatus.

A xylanolytic amyloglucosidase of Termitomyces clypeatus was characterised with respect to other amyloglucosidases. The enzyme contained high alpha-helix destabilising amino acids but no sulphur amino acid. It contained high threonine and serine, analogous to other raw starch hydrolysing enzymes. Both xylanase and amyloglucosidase activities were gradually lost with the progress of tryptophan oxidation by NBS and total inactivation occurred after oxidation of 4-5 tryptophan residues. In the presence of substrates (either starch or xylan), complete inactivation of either activities was not noticed even after oxidation of 7.7 mol of tryptophan residues. Inactivation by HNBB was not possible in the absence of any denaturant. Only 4.9 mol of tryptophan could be modified in the presence of 5 M urea which resulted in only 42% inhibition of activity. Thus modified enzyme had higher Vm/Km and lower pH optima in comparison to those of native enzyme. It was suggested that tryptophan was present at the substrate binding site and not at the active site. No such change in activity was noticed after modification of tyrosine, lysine or arginine residues. HPGPLC analysis of both dilute and concentrated enzyme solution indicated that the enzyme existed as an equilibrium mixture of protomer-oligomer. Perhaps for this reason molar mass of NAI modified enzyme appeared to be almost half of that modified by NAI in presence of substrate. Arrhenius plot of the enzyme also indicated reversible oligomerisation as a function of temperature.

The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase.

The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied. The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity. When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one. The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent. These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity. Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure. Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine. The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol. Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization.

Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site.

N-Bromosuccinimide (NBS) completely inactivated xylanases from Chainia and alkalophilic and thermophilic (AT) Bacillus with a concomittant decrease in absorption at 280 nm and with second-order rate constants of 10,500 and 5000 M-1.min-1, respectively at pH 6.0 and 25 degrees C. The kinetic analysis of inactivation indicated that one and three tryptophan residues were essential for the xylanase activity from Chainia and Bacillus, respectively. The xylanases were also inhibited by 2-hydroxy-5-nitrobenzyl bromide (HNBB). The modification of cysteine residues by p-hydroxymercurybenzoate (PHMB) and N-ethylmaleimide did not cause a loss in activity of the xylanase from Bacillus, whereas that from Chainia was completely inactivated. The kinetics of inactivation revealed the involvement of one cysteine residue for xylanase from Chainia with a second-order rate constant of 50,000 M-1.min-1. The PHMB-modified enzyme failed to show the presence of titrable -SH groups. Xylan afforded complete protection against inactivation by NBS, HNBB and PHMB, indicating the involvement of tryptophan and cysteine residues at the substrate-binding region of the enzyme.

On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies.

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.

State and reactivity of tryptophyl residues in two bacterial proteases from Sorangium sp.

The state and reactivity of tryptophyl residues in two proteolytic enzymes from Sorangium sp. were investigated by means of the following methods: spectrophotometric oxidation of tryptophans with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, and H2O2 in dioxane, optical rotatory dispersion, ultraviolet difference spectrophotometry, solvent perturbation and viscosity measurements. Out of two tryptophyl residues/molecule of alpha-lytic protease, one appears to be completely buried, while the other seems to be exposed. None of these two residues seem to be responsible for the activity of the enzyme. The beta-lytic protease undergoes an irreversible conformational transition between pH 5.0 and 3.5. Out of total four tryptophyl residues/molecule, only one is fully exposed at neutral pH. The other three are gradually exposed in the pH transition region. The degree of exposure and the dimensions of "cavities" shielding tryptophyl residues were estimated. The tryptophyl residues of of beta-lytic protease do not seem to participate in substrate binding or the active site; they are rather one of the determinants of the conformational state of the enzyme.

Bacillus cereus beta-lactamase. Reaction with N-bromosuccinimide and the properties of the product.

The effect of N-bromosuccinimide on the enzymatic activity and the conformation of a Bacillus cereus beta-lactamase (penicillin amido-beta-lactamase EC 3.5.2.6) was studied. Incubation with 10 muM N-bromosuccinimide caused over 95% decrease of the enzymatic activity within 15 min. Spectrophotometric titration with N-bromosuccinimide showed that the reaction proceeded in two steps. The half-inactivated enzyme was prepared by the reaction with N-bromosuccinimide and its properties examined. Amino acid analysis showed that the half-inactivated enzyme contained one residue of tryptophan less while other amino acid contents were similar. Neither the molecular weight nor the mobility in disc electrophoresis was changed. However, the affinity to a cephalexin-CH-Sepharose column was increased, and the Km value for cloxacillin was one-third that of the native enzyme, although that for benzylpenicillin was similar. These results indicate that a tryptophan residue sensitive to N-bromosuccinimide is essential for the maintenance of the rigid conformation and that its oxidation alters the enzyme in a manner such that a substrate with a bulky group in its side chain can form an enzyme-substrate complex more easily. In the native enzyme, the value of (f(a))(eff) (Lehrer, S.S. (1971) Biochemistry 10, 3254-3263), did not vary significantly in the absence or the presence of cloxacillin. In contrast, in the half-inactivated enzyme the presence of cloxacillin affected the conformation such that over two thirds of the tryptophyl fluorescence were accessible for quenching by KI, although about half was accessible in the absence of cloxacillin.

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme.

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.

External yeast beta-fructosidase. The role of tryptophyl residues in catalysis.

1. In native invertase at pH 4.6, 23 out of a total of 34 tryptophyl residues are "exposed" to oxidation with N-bromosuccinimide, the other residues being apparently shielded from the oxidant within the molecule. 2. Oxidation of 5-6 tryptophyl residues/molecule with N-bromosuccinimide is proportional to the complete inactivation of the enzyme, and appears to be specific for indole chromophore only. The ligand binding and fluorescence measurements indicate that the oxidation of native enzyme, up to 50% inhibition, apparently does not change the conformation and topography of enzymes surface. 3. Invertase is inhibited by diazonium-1-H-tetrazole. Since tyrosine residues can be excluded by nitration studies as catalytically unimportant, it appears that a mocification of a single histidyl residue/molecule with diazonium-1-H-tetrazole is sufficient to abolish the enzymic activity. However, the absence of inhibition with diethyl pyrocarbonate indicates that the inhibition with diazonium-1-H-tetrazole may be mediated through steric hindrance or other indirect effects. 4. The absence of inhibition with 2,4-dinitrophenylhydrazine, trinitro benzenesulfonic acid and 5,5'-dithiobis-(2-nitrobenzoate) indicates that the carbonyl groups of the carbohydrate moiety, free amino and -SH groups are not essential for activity.

Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa.

Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.

Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp.

Purine nucleotide pyrophosphotransferase was purified to apparent homogeneity from a culture filtrate of Streptomyces morookaensis. It is a monomeric protein with a molecular weight of 24 000-25 000, and its isoelectric point is 6.9. The enzyme synthesizes purine nucleoside 5'-phosphate (mono, di, or tri) 3'-diphosphates such as pppApp, ppApp, pApp, pppGpp, ppGpp and pppIpp by transferring a pyrophosphoryl group from the 5'-position of ATP, dATP and ppApp to the 3'-position of purine nucleotides. The purified enzyme catalysed the formation of 435 mumol of pppApp and 620 mumol of pppGpp from ATP and GTP per min mg protein under the standard conditions. The enzyme requires absolutely a divalent cation for activity, and optimum pH for the enzyme activity lay above 10 for Mg2+, for Co2+ and Zn2+ from 9 to 9.5, and for Fe2+ from 7.5 to 8. The following Michaelis constants were determined: AMP, 2.78 mM; ADP, 3.23 mM; GMP, 0.89 mM; GDP, 0.46 mM and GTP, 1.54 mM, in the case of ATP donor. The enzyme is inhibited by guanine, guanosine, dGDP, dGTP, N-bromosuccinimide, iodacetate, sodium borate and mercuric acetate.