The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.
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 A (1 A=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.
Kanade
Santosh R
SR
Department of Protein Chemistry and Technology, Central Food Technological Research Institute (CFTRI), Mysore-570 020, India.
Paul
Beena
B
Rao
A G Appu
AG
Gowda
Lalitha R
LR
D000143:Acids; D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000494:pharmacology; D004156:Catechol Oxidase / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D031294:Dolichos / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013696:Temperature
D000143:Acids; D002264:Carboxylic Acids; D021122:Protein Subunits; D012967:Sodium Dodecyl Sulfate; D004156:Catechol Oxidase