Mode of Action and Partial Purification of the Active Centre of exo-Poly-α-D-galacturonosidase from Selenomonas ruminantium

1993 ◽  
Vol 58 (3) ◽  
pp. 681-692
Author(s):  
Kveta Heinrichová ◽  
Július Heinrich ◽  
Mária Dzúrová ◽  
Alexander Ziolecki
Keyword(s):  
Mode Of Action ◽  
Kinetic Data ◽  
Degree Of Polymerization ◽  
Partial Purification ◽  
Catalytic Hydrolysis ◽  
Product Analysis ◽  
Active Centre ◽  
Selenomonas Ruminantium ◽  
Michaelis Constants ◽  
Hydrolysis Of

In the presented paper are summarized results of the study of the mode of action, dimensions and arrangement of the active centre of the exo-poly-α-D-galacturonosidase, (poly(1,4-α-D-galactosiduronate) digalacturonohydrolase, E.C. 3.2.1.82) produced by the bacteria Selenomonas ruminantium. With this aim we determined experimentally values of Michaelis constants and limiting rates for the catalytic hydrolysis of linear oligo(D-galactosiduronates) of the degree of polymeration in the range of 3 to 8, at pH 7.0 and the temperature 30 °C. We calculated molecular activities k0 and parameters k0/Km from these values. From the dependence of logk0 and logk0/Km on the degree of polymerization five subsites of the active centre were determined, with the catalytic site being situated between the second and third one. Kinetic data were used for the calculation of the affinities of the fourth and fifth subsites A4 and A5 in accordance with the theory of Hiromi. Product analysis of non-labelled oligo(D-galactosiduronates) and compounds labelled with [1-3H] at the reducing end anabled to ascertain approximately the value for the first subsite A1 of the active centre and to study the mode of action of the exo-poly-α-D-galacturonosidase from Selenomonas ruminantium.

scholarly journals Relationship of Exo-β-d-Galactofuranosidase Kinetic Parameters to the Number of Phosphodiesters in Penicillium fellutanum Peptidophosphogalactomannan: Enzyme Purification and Kinetics of Glycopeptide and Galactofuran Chain Hydrolysis

2001 ◽  
Vol 67 (10) ◽  
pp. 4648-4656 ◽  
Author(s):  
Brigitte A. Tuekam ◽  
Yong-Il Park ◽  
Clifford J. Unkefer ◽  
John E. Gander
Keyword(s):  
Kinetic Parameters ◽  
Kinetic Data ◽  
Inorganic Phosphate ◽  
Enzyme Purification ◽  
Degree Of Polymerization ◽  
Apparent Homogeneity ◽  
Relationship Of ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACT Extracellular Penicillium fellutanumexo-β-d-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP2GMii and pP25GMii(containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-β-d-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP2GMii or pP25GMii. Thek cat/Km value for pP25GMii is 1.7 × 103M−1 s−1, that for 1-O-methyl-β-d-galactofuranoside is 1.1 × 104 M−1 s−1, that for pP2GMii is 1.7 × 10 4M−1 s−1, and those for 5-O-β-d-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 105 and 4.1 × 105 M−1s−1, respectively. Variability in thek cat/Km values is due primarily to differences in Km values; thek −1/k 1 ratio likely provides the most influence on Km. k cat increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pPxGMii added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml−1h−1. No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pPxGMii and of related polymers is proportional to thek cat/Km value of each polymer. The kinetic data show that thek cat/Km value increases as the number of phosphodiesters of pPxGMiidecreases, also resulting in an increase in the activity of exo-β-d-galactofuranosidase.

scholarly journals Study of the mode of action and site-specificity of the endo-(1→4)-β-d-glucanases of the fungus Penicillium pinophilum with normal, 1-3H-labelled, reduced and chromogenic cello-oligosaccharides

1990 ◽  
Vol 266 (2) ◽  
pp. 371-378 ◽  
Author(s):  
K M Bhat ◽  
A J Hay ◽  
M Claeyssens ◽  
T M Wood
Keyword(s):  
Cell Membrane ◽  
Mode Of Action ◽  
Analytical Data ◽  
Degree Of Polymerization ◽  
The Other ◽  
Site Specificity ◽  
Glycosidic Bonds ◽  
Penicillium Pinophilum ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

The modes of action of the five major endo-(1→4)-beta-D-glucanases (I, II, III, IV and V) purified from Penicillium pinophilum cellulase were compared by h.p.l.c. analysis, with normal, 1-3H-labelled and reduced cello-oligosaccharides and 4-methylumbelliferyl glycosides as substrates. Significant differences were observed in the preferred site of cleavage even when substrates with the same number of glycosidic bonds were compared. Thus, although endoglucanase I was unable to attack normal cello-oligosaccharides shorter than degree of polymerization 6, it hydrolysed reduced cellopentaose to yield cellotriose and cellobi-itol, and it produced cellotriose and 4-methylumbelliferyl glucoside from 4-methylumbelliferyl cellotetraoside. Endoglucanase IV hydrolysed [1-3H]cellotriose but did not attack either cellotri-itol or 4-methylumbelliferyl cellobioside. These and other anomalous results indicated clearly that modification of the reducing glycosyl residue on the cello-oligosaccharides induces in an apparent change in the mode of action of the endoglucanases. It is suggested that, although cello-oligosaccharide derivatives are useful for differentiating and classifying endoglucanases, conclusions on the mechanism of cellulase action resulting from these measurements should be treated cautiously. Unequivocal information on the mode of endoglucanase action on cello-oligosaccharides was obtained with radiolabelled cello-oligosaccharides of degree of polymerization 3 to 5. Indications that transglycosylation was a property of the endoglucanases were particularly evident with the 4-methylumbelliferyl cello-oligosaccharides. Turnover numbers for hydrolysis of the umbelliferyl cello-oligosaccharides were calculated, and these, along with the other analytical data collected on the products of hydrolysis of the normal, reduced and radiolabelled cello-oligosaccharides, suggested that the various endoglucanases had different roles to play in the overall hydrolysis of cellulose to sugars small enough to be transported through the cell membrane.

scholarly journals Hydrolysis kinetics of p-nitrophenylpicolinate catalyzed by schiff base Mn(III) complexes in Gemini 16-2-16 micellar solutiony

2009 ◽  
Vol 7 (3) ◽  
pp. 542-549 ◽  
Author(s):  
Bin Xu ◽  
Weidong Jiang ◽  
Jin Zhang ◽  
Guangyin Fan ◽  
Jianzhang Li
Keyword(s):  
Schiff Base ◽  
Micellar Solution ◽  
Cetyltrimethylammonium Bromide ◽  
Complex Structures ◽  
Catalytic Hydrolysis ◽  
Enzyme Mimics ◽  
Active Centre ◽  
L1 And L2 ◽  
Kinetics Of ◽  
Hydrolysis Of

AbstractTwo mono-Schiff base Mn(III) complexes (MnL2Cl, L=L1 and L2) were synthesized and employed as artificial hydrolases in catalyzing the hydrolysis of p-nitrophenylpicolinate (PNPP) in Gemini 16-2-16 micellar solution. The effect of different micelles and their complex structures on the catalytic hydrolysis of PNPP is discussed in detail. The observations showed that MnL 22Cl exhibited higher catalytic activity over MnL 12Cl under a comparable condition, which confirmed that an open active centre is essential for modulating the activities of the two enzyme mimics. Moreover, under the conditions employed, hydrolytic rates of PNPP induced by these Mn(III) complexes were faster in Gemini 16-2-16 micelles than that in the micellar solution of cetyltrimethylammonium bromide (CTAB), a conventional analogue of Gemini 16-2-16.

Partial Characterization of the Active Site of Carrot Poly(galacturonate) Hydrolase and Degradation of Oligo(D-galactosiduronates)

1995 ◽  
Vol 60 (2) ◽  
pp. 328-338 ◽  
Author(s):  
Kveta Heinrichová ◽  
Július Heinrich ◽  
Mária Dzúrová
Keyword(s):  
Active Center ◽  
Hydrolytic Cleavage ◽  
Catalytic Hydrolysis ◽  
Optimal Conditions ◽  
Polymerization Degree ◽  
Enzyme Degradation ◽  
Good Accord ◽  
Michaelis Constants ◽  
Hydrolysis Of

The dimension and arrangement of the active center in carrot poly(1→4-α-D-galacturonide)galacturonohydrolase (E.C.3.2.1.67) is studied. Molecular activities k0 and parameters k0/Km were calculated from the experimentally determined Michaelis constants Km and maximum rates of catalytic hydrolysis of linear oligo(D-galactosiduronates) (polymerization degree n = 2 to 7) at pH 5.0 and 30 °C. From the dependence of log k0 and log k0/Km on n we derived that the active center consists of six subsites, the catalytic site being situated between the first and second subsite. In accord with the theory of Hiromi [Hiromi K.: Biochem. Biophys. Res. Commun. 40, 1 (1970)], the kinetic data were used for calculation of affinities (Ai) of the third (A3) to the sixth (A6) subsite. Two possible models were studied for the action of carrot poly(galacturonate)hydrolase which catalyzes the gradual terminal hydrolytic cleavage of oligo(D-galactosiduronates) from the non-reducing end of the molecule. The distribution of products in monomolecular hydrolysis of penta(D-galactosiduronate) under optimal conditions (pH and temperature) indicates a multi-chain enzymatic attack with predominant single collision. The kinetic results of the enzyme degradation are in good accord with the above-mentioned assumption.

Factor V from Human Plasma

1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Kinetic Data ◽  
Human Factor ◽  
Kinetic Studies ◽  
Partial Purification ◽  
Factor V ◽  
Two Stage ◽  
Plasma Factor ◽  
Tissue Thromboplastin

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).

scholarly journals Catalytic hydrolysis of s-triazine compounds over Al2O3

1996 ◽  
Vol 27 (1-2) ◽  
pp. 167-173 ◽  
Author(s):  
Zhaoqi Zhan ◽  
Martin Müllner ◽  
Johannes A. Lercher
Keyword(s):  
Catalytic Hydrolysis ◽  
Hydrolysis Of

Catalytic Hydrolysis of 4-Nitrophenyl Phosphate by Lanthanum(III)-Hectorite

Langmuir ◽  
2003 ◽  
Vol 19 (6) ◽  
pp. 2188-2192 ◽  
Author(s):  
Steven T. Frey ◽  
Benjamin M. Hutchins ◽  
Brian J. Anderson ◽  
Teresa K. Schreiber ◽  
Michael E. Hagerman
Keyword(s):  
Catalytic Hydrolysis ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of
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