scholarly journals The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

scholarly journals Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus

1972 ◽  
Vol 128 (2) ◽  
pp. 243-252 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Original Activity ◽  
Solubilized Enzyme ◽  
High Concentrations

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-α-d-mannose was used as a substrate was a β-(1→4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent Km is 55–62μm, and a disproportionate increase in rate is observed at high concentrations. GDP-α-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent Ki of 6.2μm. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0°C or in 24h at −20°C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at −20°C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.

scholarly journals Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

1997 ◽  
Vol 326 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Shoshana KEYNAN ◽  
Nigel M. HOOPER ◽  
Anthony J. TURNER
Keyword(s):  
Specific Activity ◽  
Mammalian Species ◽  
Competitive Inhibitor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Renal Metabolism ◽  
Leukotriene D4 ◽  
Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

scholarly journals (335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1083C-1083
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations ◽  
Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

scholarly journals Partial purification and properties of l-asparagine synthetase from mouse pancreas

1979 ◽  
Vol 181 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Harry A. Milman ◽  
David A. Cooney
Keyword(s):  
Specific Activity ◽  
Asparagine Synthetase ◽  
Partial Purification ◽  
Synthetase Activity ◽  
Mouse Pancreas ◽  
Purification And Properties ◽  
Bean Trypsin Inhibitor ◽  
Thiol Reagent ◽  
High Concentrations ◽  
Glutaminase Activity

l-Asparagine synthetase was partially purified from mouse pancreas to a final mean specific activity of 0.10 unit/mg of protein. The enzyme exhibited an l-glutaminase activity which was not affected by l-asparate, NH4Cl, ATP–MgCl2, l-glutamate, AMP (sodium salt) or sodium pyrophosphate. The l-glutamine-dependent l-asparagine synthetase activity of the partially purified enzyme from mouse pancreas was markedly decreased by freezing for 7 days at −87°C in the presence of 1mm-dithiothreitol, but effectively protected from inactivation by high concentrations (10mm) of the thiol reagent. The l-glutaminase activity of the enzyme was inhibited by antagonists of l-glutamine (e.g. 6-diazo-5-oxo-l-norleucine, 5-chloro-4-oxo-l-norvaline, 5-diazo-4-oxo-l-norvaline and NSC-163501) and thiol-reactive compounds (e.g. 2-amino-4-arsenophenol hydrochloride, maleimide, mucochloric acid and ZnCl2), but not by aminomalonic acid, the next lower homologue of l-aspartate, nor by l-homoserine β-adenylate, an analogue of the presumed transitory covalent intermediate. The complete forward reaction catalysed by l-asparagine synthetase from mouse pancreas appears to be irreversible and essentially stoicheiometric under the conditions examined. Mouse pancreas contains a proteolytic inhibitor of l-asparagine synthetase separable from the enzyme by ion-exchange column chromatography. The inhibitor is activated by incubation at 4°C for 110h and inactivated by soya-bean trypsin inhibitor, di-isopropyl phosphorofluoridate and boiling.

scholarly journals Partial purification of a diacylglycerol lipase from bovine aorta

1994 ◽  
Vol 298 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M W Lee ◽  
D L Severson
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Short Chain ◽  
Partial Purification ◽  
Diacylglycerol Lipase ◽  
Bivalent Cations ◽  
Triton X ◽  
Hydrolysis Of ◽  
Long Chain

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties

1988 ◽  
Vol 66 (2) ◽  
pp. 148-157 ◽  
Author(s):  
Félix Busto ◽  
Pilar Del Valle ◽  
Joaquín Soler
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Kinetic Properties ◽  
Saturation Curve ◽  
Phycomyces Blakesleeanus ◽  
Competitive Inhibitors ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) was purified approximately 500-fold to a final specific activity of 25 U∙mg protein−1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces requires Mg2+ for activity, but not K+ or NH4. It showed a transition temperature at 36 °C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curves to a hyperboolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type enzyme.

scholarly journals Mutagenesis of residue 157 in the active site of human glyoxalase I

1997 ◽  
Vol 328 (1) ◽  
pp. 231-235 ◽  
Author(s):  
Marianne RIDDERSTRÖM ◽  
D. Alexander CAMERON ◽  
T. Alwyn JONES ◽  
Bengt MANNERVIK
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Glyoxalase I ◽  
Wild Type ◽  
Direct Role ◽  
Mutant Proteins ◽  
Competitive Inhibitors ◽  
Km Value

Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis. The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel. The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme. The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx. 20% of wild-type activity. The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value. This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant. With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I. Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values. The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I. The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.

The Activator-Binding Site of Onchocerca volvulus S-Adenosylmethionine Decarboxylase, a Potential Drug Target

2003 ◽  
Vol 384 (8) ◽  
pp. 1195-1201 ◽  
Author(s):  
D. Ndjonka ◽  
Y. Zou ◽  
X. Bi ◽  
P. Woster ◽  
R. D. Walter ◽  
...  
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Onchocerca Volvulus ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Polyamine Analogues ◽  
Human Enzyme ◽  
Competitive Inhibitors ◽  
New Strategy ◽  
Key Enzyme

Abstract S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. In many eukaryotes its activity is stimulated specifically by putrescine. The AdoMetDC of the filarial parasite Onchocerca volvulus, however, is not only stimulated by putrescine but also by the naturally occuring polyamines spermidine and spermine. Several diamines, acetylated polyamines and polyamine analogues were used to analyse what molecular prerequisites are needed to stimulate nematode AdoMetDC activity. In the absence of an activator, the O. volvulus enzyme exhibits an extremely low specific activity. This fact, together with the unspecificity of activator binding, was thought to be useful for a new strategy to inhibit nematode AdoMetDC activity. Therefore, different polyamine analogues were tested as competitive inhibitors towards the stimulatory effect putrescine has on the O. volvulus and, in comparison, on the Caenorhabditis elegans and human AdoMetDC. Bis(aralkyl)- and bis(alkyl)-substituted polyamine analogues with a 3-7-3 backbone were found to inhibit AdoMetDC activities, however, probably without interfering with the putrescine stimulation. The best inhibitor, BW-1, was about 10-fold more effective against O. volvulus AdoMetDC than against the human enzyme. Unexpectedly, BW-1 was determined to be a competitive inhibitor with respect to AdoMet, having a Ki value of 310 uM for the putrescine-stimulated human AdoMetDC. Furthermore, we show for the O. volvulus and the human enzyme that the degree of inhibition by BW-1 depends on the actual putrescine concentration.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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