scholarly journals The purification and properties of a β-N-acetylhexosaminidase from Trichomonas foetus

1975 ◽  
Vol 151 (1) ◽  
pp. 145-148 ◽  
Author(s):  
R G Edwards ◽  
P Thomas ◽  
J H Westwood
Keyword(s):  
Affinity Chromatography ◽  
Active Site ◽  
Substrate Binding ◽  
Hydroxyl Group ◽  
Ph Optimum ◽  
Dual Specificity ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Catalytic Rate

A β-N-acetylhexosaminidase was purified 800-fold from extracts of Trichomonas foetus by affinity chromatography on a column of N-(epsilon-aminohexanoyl)-2-acetamido-2-deoxy-β-D-glucopyranosylamine bound to CNBr-activated Sepharose. The enzyme has a dual specificity for the p-nitrophenyl β-D-glycosides of N-acetylglucosamine and N-acetyl-galactosamine. The parent sugars are both competitive inhibitors. The enzyme has a mol. wt. approx. 150000 and a pH optimum of 6.2. It is suggested that the same active site catalyses both activities and that no part is played by the 4-hydroxyl group in substrate binding, but it is involved in determining the catalytic rate.

scholarly journals Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

1978 ◽  
Vol 175 (2) ◽  
pp. 743-750 ◽  
Author(s):  
P Calvo ◽  
A Reglero ◽  
J A Cabezas
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mixed Substrates ◽  
Ph Optimum ◽  
Terrestrial Mollusc ◽  
Buffer Solutions ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Ph Range

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8–4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

scholarly journals Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

2010 ◽  
Vol 66 (6) ◽  
pp. 673-684 ◽  
Author(s):  
Radhika Malik ◽  
Ronald E. Viola
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Substrate Binding ◽  
Hydride Transfer ◽  
Divalent Metal ◽  
Ammonium Ion ◽  
Hydroxyl Group ◽  
Metal Ion Binding ◽  
Cofactor Binding ◽  
Divalent Metal Ion

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+and NADH. This TDH structure fromPseudomonas putidahas a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

scholarly journals Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase

1974 ◽  
Vol 144 (3) ◽  
pp. 513-517 ◽  
Author(s):  
R L Robson ◽  
J G Morris
Keyword(s):  
Energy Charge ◽  
Clostridium Pasteurianum ◽  
Adenylate Energy Charge ◽  
Ph Optimum ◽  
Exogenous Glucose ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Nucleotide Sugars

1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized α-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, Km for Pi=2.2mm, and (b) in the synthetic direction, Km for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (Ki=20μm) and UDP-glucose (Ki=60μm) were particularly potent competitive inhibitors. ATP, NADP+and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ‘glucose excess’ chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.

Atomic resolution structure of a lysine-specific endoproteinase fromLysobacter enzymogenessuggests a hydroxyl group bound to the oxyanion hole

2014 ◽  
Vol 70 (7) ◽  
pp. 1832-1843 ◽  
Author(s):  
Peter Asztalos ◽  
Astrid Müller ◽  
Werner Hölke ◽  
Harald Sobek ◽  
Markus G. Rudolph
Keyword(s):  
Serine Protease ◽  
Binding Sites ◽  
Substrate Binding ◽  
High Specificity ◽  
Catalytic Triad ◽  
Hydroxyl Group ◽  
Ph Optimum ◽  
Oxyanion Hole ◽  
High Ph ◽  
Substrate Binding Sites

Lysobacter enzymogeneslysyl endoproteinase (LysC) is a trypsin-type serine protease with a high pH optimum that hydrolyses all Lys-Xaa peptide bonds. The high specificity of LysC renders it useful for biotechnological purposes. The K30R variant of a related lysyl endoproteinase fromAchromobacter lyticushas favourable enzymatic properties that might be transferrable to LysC. To visualize structural differences in the substrate-binding sites, the crystal structures of wild-type and the K30R variant of LysC were determined. The mutation is located at a distance of 12 Å from the catalytic triad and subtly changes the surface properties of the substrate-binding site. The high pH optimum of LysC can be attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate and is a general feature of this enzyme subfamily. LysC crystals in complex with the covalent inhibitorNα-p-tosyl-lysyl chloromethylketone yielded data to 1.1 and 0.9 Å resolution, resulting in unprecedented precision of the active and substrate-binding sites for this enzyme subfamily. Error estimates on bond lengths and difference electron density indicate that instead of the expected oxyanion a hydroxyl group binds to the partially solvent-exposed oxyanion hole. Protonation of the alkoxide catalytic intermediate might be a recurring feature during serine protease catalysis.

scholarly journals Purification and properties of 3′-nucleotidase of Leishmania donovani

1992 ◽  
Vol 285 (1) ◽  
pp. 41-46 ◽  
Author(s):  
G O Gbenle ◽  
D M Dwyer
Keyword(s):  
Leishmania Donovani ◽  
Intact Cell ◽  
Surface Membrane ◽  
Low Latency ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
Ph Values ◽  
Purification And Properties ◽  
Sds Page ◽  
Competitive Inhibitors

A surface membrane 3′-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3′-phosphates. 3′-AMP and 3′-IMP are the best substrates, whereas ADP, ATP, 2′-deoxyadenosine 3′-phosphate and 5′-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3′-nucleotides of the parasite's host.

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

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