scholarly journals The active site of β-glucosidase from Botryodiplodia theobromae. Effects of pH and dioxan on enzyme-catalysed reactions

1977 ◽  
Vol 167 (3) ◽  
pp. 831-833 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Maximum Velocity ◽  
Carboxyl Group ◽  
Michaelis Constant ◽  
Botryodiplodia Theobromae ◽  
Effects Of Ph ◽  
Group 2 ◽  
Beta Glucosidase ◽  
Hydrolysis Of

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat in the absence or presence of added dioxan was found to be dependent on the ionization of two groups, which appeared to be a carboxyl group and an imidazole group. 2. Dioxan increased the Michaelis constant, Km, but decreased the maximum velocity, V.

scholarly journals The β-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions

1979 ◽  
Vol 179 (3) ◽  
pp. 503-507 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Maximum Velocity ◽  
Enzymic Activity ◽  
Enzyme Mechanism ◽  
Botryodiplodia Theobromae ◽  
Imidazole Group ◽  
Group 4 ◽  
Effects Of Ph ◽  
Group 3 ◽  
High Concentrations ◽  
Beta Glucosidase

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45–46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.

scholarly journals The active site of penicillinase from Staphylococcus aureus PC1. Isolation of a specific covalent complex with the substrate quinacillin

1975 ◽  
Vol 149 (2) ◽  
pp. 397-401 ◽  
Author(s):  
R Virden ◽  
A F Bristow ◽  
R H Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Dodecyl Sulphate ◽  
Active Site ◽  
Sodium Dodecyl ◽  
Carboxyl Group ◽  
Guanidinium Chloride ◽  
Group 3 ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Covalent Complex

1. The penicillinase-catalysed hydrolysis of quinacillin was quenched by addition of 5 m-guanidinium chloride or 1% (w/v) sodium dodecyl sulphate, and the quenched reaction mixture was dialysed exhaustively against solutions of the denaturant. 2. Irreversibly bound quinacillin was shown by titration with HgCl2 to be covalently attached to the protein by the β-lactam carboxyl group. 3. The derivative was found to be stable over the pH range 3.5-8.5. 4. Chymotryptic hydrolysis of the product and subsequent fractionation showed that quinacillin was bound to one or possibly two peptides.

scholarly journals Significant Role of Fragment 1·2 Plus Light Chain of Bovine High Molecular Weight Kininogen in Contact Mediated Coagulation

1977 ◽  
Author(s):  
R. Waldmann ◽  
A. G. Scicli ◽  
G. M. Scicli ◽  
J. A. Guimaraes ◽  
O. A. Carretero ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Light Chain ◽  
Active Site ◽  
Urinary Kallikrein ◽  
High Molecular Weight Kininogen ◽  
Molar Basis ◽  
Hydrolysis Of ◽  
Effect Of Light

We have previously reported that high molecular weight kininogen (HMWK) partially corrects the partial thromboplastin time (aPTT) of Fitzgerald Trait plasma (HMWK deficiency), and in addition, we have also reported that kinin free HMWK which is also Fragment 1·2 free has decreased correcting capability. Presently, we have studied the effect of lysyl-bradykinin-free HMWK with Fragment 1·2 intact, obtained by hydrolysis of HMWK with urinary kallikrein. In addition, the effect of light chain with and without attached Fragment 1·2 were studied. When the correcting capacity of HMWK was compared, on a molar basis with the above-mentioned fragments, the following results were obtained. Kinin and Fragment 1.2 free HMWK has a significantly decreased correcting capacity on the aPTT of Fitzgerald trait plasma while lysyl-bradykinin free HMWK shows no decrease in correcting capability. Light chain with Fragment 1·2 attached also has intact correcting capability while light chain alone has a significantly decreased correcting activity. The above data suggest that the active site of HMWK resides in the light chain with Fragment 1·2 attached. Since Fragment 1·2 alone has inhibitory capacity, it may serve as a binding site and the light chain as the active site.

Purification et quelques propriétés de l'AMPc phosphodiestérase d'Arthrobacter crystallopoietes

1988 ◽  
Vol 66 (5) ◽  
pp. 454-459 ◽  
Author(s):  
François Schneider ◽  
Gérard Lefebvre ◽  
Michèle Ribolzi-Chery ◽  
Jean-Michel Bertin ◽  
Robert Gay
Keyword(s):  
Molecular Weight ◽  
Active Site ◽  
Cyclic Nucleotides ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Negative Cooperativity ◽  
Divalent Ions ◽  
Camp Phosphodiesterase ◽  
Hydrolysis Of

cAMP phosphodiesterase was purified 8250-fold from extracts of Arthrobacter crystallopoietes, primarily by hydrophobic chromatography. The molecular weight of this enzyme was estimated as 51 000 by gel filtration and density-gradient centrifugation. The results suggest that the enzyme consists of two subunits with a molecular weight of 25 600. Properties of this enzyme are reported, including its negative cooperativity. This phosphodiesterase specifically catalyzes the hydrolysis of 3′,5′-cyclic nucleotides. Divalent ions either have no effect on activity or are weak inhibitors. Photooxidation of the enzyme with methylene blue and treatment with mercuribenzoates suggest that this enzyme may possess an imidazole group within its active site. The effects of thiols and Fe2+ on activity suggests that this enzyme may be a metalloenzyme.

Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus faecalis

1988 ◽  
Vol 66 (1) ◽  
pp. 76-80 ◽  
Author(s):  
E. B. Waygood ◽  
K. Pasloske ◽  
L. T. J. Delbaere ◽  
J. Deutscher ◽  
W. Hengstenberg
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Phosphotransferase System ◽  
Temperature Dependent ◽  
Streptococcus Faecalis ◽  
Terminal Residue ◽  
Hydrolysis Of

The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1- phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.

scholarly journals The β-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action

1981 ◽  
Vol 199 (1) ◽  
pp. 203-209 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Isotope Effect ◽  
Maximum Velocity ◽  
Competitive Inhibitor ◽  
Ion Pair ◽  
High Concentration ◽  
Botryodiplodia Theobromae ◽  
Solvent Isotope Effect ◽  
Kinetic Isotope ◽  
Beta Glucosidase ◽  
Solvent Isotope

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.

Porcine Liver Esterase-Catalyzed Hydrolysis of Methyl Tri-O-acetyl-β-D-arabinopyranoside, Methyl Tri-O-acetyl-β-D-ribopyranoside and Methyl Tri-O-acetyl-β-D-ribofuranoside

2000 ◽  
Vol 65 (10) ◽  
pp. 1619-1629 ◽  
Author(s):  
Jitka Moravcová ◽  
Karel Kefurt ◽  
Romana Hladůvková ◽  
Jan Staněk
Keyword(s):  
Active Site ◽  
Michaelis Constant ◽  
Porcine Liver ◽  
Site Model ◽  
Liver Esterase ◽  
Preparative Yield ◽  
Active Site Model ◽  
Catalyzed Reactions ◽  
Hydrolysis Of ◽  
Porcine Liver Esterase

Methyl 2,3,4-tri-O-acetyl-β-D-arabinopyranoside (1), methyl 2,3,4-tri-O-acetyl-β-D-ribopyranoside (2), and methyl 2,3,5-tri-O-acetyl-β-D-ribofuranoside (3) were deacetylated in porcine liver esterase-catalyzed reactions. Triacetate 1 gave methyl 3,4-di-O-acetyl-β-D-arabinopyranoside in 70% preparative yield while the regioselectivities found for the substrates 2 and 3 were substantially lower. Both the Michaelis constant and maximum rate were calculated for deacetylation of 1, 2, and 3. The results were interpreted using an active site model for the esterase proposed by Jones.

scholarly journals A New Synthetic Route toN-Benzyl Carboxamides through the Reverse Reaction ofN-Substituted Formamide Deformylase

2013 ◽  
Vol 80 (1) ◽  
pp. 61-69
Author(s):  
Yoshiteru Hashimoto ◽  
Toshihide Sakashita ◽  
Hiroshi Fukatsu ◽  
Hiroyoshi Sato ◽  
Michihiko Kobayashi
Keyword(s):  
Active Site ◽  
Reverse Reaction ◽  
Synthetic Route ◽  
Forward Reaction ◽  
Enzyme Active Site ◽  
Dead End ◽  
Effects Of Ph ◽  
Inhibition Studies ◽  
Hydrolysis Of ◽  
Substrate Concentrations

ABSTRACTPreviously, we isolated a new enzyme,N-substituted formamide deformylase, that catalyzes the hydrolysis ofN-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726–13731, 2004,doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizingN-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C1to C3), were active as acid substrates for the reverse reaction. Through this reaction,N-substituted carboxamides, such as NBFA,N-benzylacetamide, andN-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates.

scholarly journals The β-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl β-d-glucopyranoside in dioxan/water

1978 ◽  
Vol 175 (2) ◽  
pp. 455-459 ◽  
Author(s):  
G M Umezurike
Keyword(s):  
Maximum Rate ◽  
Buffer System ◽  
Botryodiplodia Theobromae ◽  
Hydrolytic Reaction ◽  
Anomeric Carbon ◽  
Rate Of Hydrolysis ◽  
Beta Glucosidase ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Anomeric Carbon Atom

1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Botryodiplodia theobromae Pat. has been studied in the presence of added dioxan. 2. At donor saturation, the maximum rate of hydrolysis in the presence of up to 50%(v/v) dioxan was pH4.3-4.5 (pH of the buffer system in water) in McIlvaine's buffer. 3. Increasing dioxan concentrations progressively decreased the maximum rate of hydrolysis. 4. The rate of enzyme-catalysed reaction was enhanced at high donor concentrations, but inhibited at low donor concentrations in the presence of glycerol, methanol, fructose of sucrose. 5. The hydrolytic reaction was found to proceed with retention of configuration at the anomeric carbon atom. 6. The kinetics of the enzyme-catalysed process in the presence of added acceptors indicated that water was necessary for the maintenance of the active enzyme conformation apart from its acceptor function.

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