Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms

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 coexis ting and mutually interconvertible isomers of different specific activity, a phenomenon that has been termed microheterogeneity and for which a struct ural explanation has not yet been presented. The enzyme can be activated by reduction, in particular by thiol reagents, and inactivated by oxidation a nd the concomitant change of the patterns of its isomeric forms could be qu antified 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 wa s obtained. This form was almost inaccessible for proteolysis by trypsin wh ile the oxidized and GSH-activated enzyme yielded four peptides when treate d with trypsin. Their sequence indicated cleavage predominantly of loops co nnecting the P-strands and a-helices of the (beta/alpha)(8)-barrel which fo rms 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 p ullulanase. In vivo, the enzyme maintains its activated state due to the hi gh concentration of GSH in the chloroplast. However, the chloroplast's pH s hifts from day (pH 8) to night (pH 7) and thus could also alter the activit y of the protein in accordance with the required function in starch metabol ism. (C) 2001 Elsevier Science B.V. All rights reserved.