title: Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms.
abstract: Spinach starch debranching enzyme, a limit dextrinase or pullulanase (EC 3.2.1.41), is a monomeric protein of 100 kDa that produces up to seven coexisting and mutually interconvertible isomers of different specific activity, a phenomenon that has been termed microheterogeneity and for which a structural explanation has not yet been presented. The enzyme can be activated by reduction, in particular by thiol reagents, and inactivated by oxidation and the concomitant change of the patterns of its isomeric forms could be quantified by chromatofocusing. The hypothesis was examined that reduction of the enzyme's thiol groups shifts the isomer pattern towards the forms with a higher specific activity while oxidation favours the less active forms. Using TCEP as reductant only the form with the highest specific activity was obtained. This form was almost inaccessible for proteolysis by trypsin while the oxidized and GSH-activated enzyme yielded four peptides when treated with trypsin. Their sequence indicated cleavage predominantly of loops connecting the beta-strands and alpha-helices of the (beta/alpha)(8)-barrel which forms the catalytic site of the pullulanase. Formation of various disulphide bridges between the loops connecting the barrel structures -- predominantly on one side -- may be the reason for the microheterogeneity of the spinach pullulanase. In vivo, the enzyme maintains its activated state due to the high concentration of GSH in the chloroplast. However, the chloroplast's pH shifts from day (pH 8) to night (pH 7) and thus could also alter the activity of the protein in accordance with the required function in starch metabolism.
authors.affiliation: Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Schindler
authors.forename: A
authors.initials: A
authors.lastname: Renz
authors.forename: F X
authors.initials: FX
authors.lastname: Schmid
authors.forename: E
authors.initials: E
authors.lastname: Beck
mesh_terms: D002736:Chloroplasts / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D018724:Spinacia oleracea / Q000201:enzymology*; D014357:Trypsin
chemical_list: D007527:Isoenzymes; D006026:Glycoside Hydrolases; C100181:pullulanase; D014357:Trypsin; D005978:Glutathione