scholarly journals The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

2006 ◽  
Vol 395 (3) ◽  
pp. 551-562 ◽  
Author(s):  
Santosh R. Kanade ◽  
Beena Paul ◽  
A. G. Appu Rao ◽  
Lalitha R. Gowda
Keyword(s):  
Polyphenol Oxidase ◽  
Insect Herbivory ◽  
Catalytic Efficiency ◽  
Critical Micellar Concentration ◽  
Field Bean ◽  
Ph Optimum ◽  
Dolichos Lablab ◽  
Acid Ph ◽  
Stokes Radius ◽  
Increased Sensitivity

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.

The unique enzymatic function of field bean (Dolichos lablab) d-galactose specific lectin: a polyphenol oxidase

2008 ◽  
Vol 26 (5) ◽  
pp. 535-545 ◽  
Author(s):  
Santosh R. Kanade ◽  
Devavratha H. Rao ◽  
Ramanath N. Hegde ◽  
Lalitha R. Gowda
Keyword(s):  
Polyphenol Oxidase ◽  
Field Bean ◽  
Dolichos Lablab ◽  
Enzymatic Function

scholarly journals G-6-PD Long Prairie: a new glucose-6-phosphate dehydrogenase mutant exhibiting normal sensitivity to inhibition by NADPH and accompanied by nonspherocytic hemolytic anemia

Blood ◽  
1977 ◽  
Vol 49 (2) ◽  
pp. 247-251 ◽  
Author(s):  
GJ Johnson ◽  
ME Kaplan ◽  
E Beutler
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Alternative Mechanism ◽  
Ph Optimum ◽  
Intracellular Enzyme ◽  
Chronic Hemolysis ◽  
Increased Sensitivity ◽  
Normal Sensitivity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Heat Instability

Abstract The enzymatic properties of a new glucose-6-phosphate dehydrogenase (G- 6-PD) variant (G-6-PD Long Prairie) were studied in a white patient with chronic nonspherocytic hemolysis. The red cells were found to have 2.3%-7.7% normal enzymatic activity. The mutant enzyme exhibited marked heat instability, an increased pH optimum, a moderately decreased Km for G-6-P, and increased utilization of 2-deoxyglucose-6-phosphate and deamino NADP. The Km for NADP and Ki for NADPH were both normal. G-6-PD Long Prairie is an interesting new G-6-PD variant that demonstrates that chronic hemolysis can be associated with modestly decreased G-6-PD activity despite normal sensitivity to inhibition by NADPH. Although increased sensitivity to inhibition by NADPH has been postulated to decrease intracellular enzyme activity, resulting in enhanced susceptibility to hemolysis in certain G-6-PD variants with only moderately decreased enzymatic activity, an alternative mechanism of hemolysis, possibly enzyme thermolability, exists in G-6-PD Long Prairie.

scholarly journals PREPARATION AND CHARACTERIZATION OF AN IMMUNOELECTRON MICROSCOPE TRACER CONSISTING OF A HEME-OCTAPEPTIDE COUPLED TO Fab

1974 ◽  
Vol 139 (1) ◽  
pp. 208-223 ◽  
Author(s):  
J. P. Kraehenbuhl ◽  
R. E. Galardy ◽  
J. D. Jamieson
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Antibody Fragments ◽  
Amino Groups ◽  
Electron Microscope Level ◽  
Ph Optimum ◽  
Peroxidatic Activity ◽  
Stokes Radius ◽  
Trypsin Hydrolysis

A heme-octapeptide (mol wt 1,550) has been obtained from cytochrome c by successive pepsin and trypsin hydrolysis and purified by gel filtration and countercurrent distribution. It possesses peroxidatic activity characterized by an apparent Km of 0.2 M, an apparent vmax of 4 mmol/min per mg of peptide, and a pH optimum of 7.0. Using a novel two-step conjugation procedure, the heme-octapeptide was coupled to rabbit Fab antibody fragments by first derivatizing it with the N-hydroxysuccinimide ester of p-formylbenzoic acid and subsequently allowing it to form a Schiff base with the amino groups of Fab. Stable covalent linkages were then obtained by reduction of the Schiff bases with sodium borohydride. The conjugate consists of ∼2 heme-octapeptides attached to each Fab molecule. The molecular weight is 45,000 daltons when coupled to sheep Fab and 50,000 daltons with a Stokes radius of 32 Å, when conjugated to rabbit Fab. Its peroxidatic activity is characterized by an apparent Km of 0.4 M, an apparent vmax of 0.4 mmol/min and per mg of attached heme-octapeptide and a pH optimum of 7.0. The conjugate has been used for the localization at the electron microscope level of secretory immunoglobulins in the mammary gland of lactating rabbits.

scholarly journals Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (7) ◽  
pp. 2230-2240 ◽  
Author(s):  
Xiaoyun Su ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Ph Optimum ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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