ROLE OF METHIONINE IN THE ACTIVE-SITE OF ALPHA-GALACTOSIDASE FROM TRICHODERMA-REESEI

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 sho ws a 12-fold increase in activity towards p-nitrophenyl alpha-D-galact opyranoside. A similar effect is produced by the treatment of alpha-ga lactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH . Galactose (a competitive inhibitor of alpha-galactosidase; K-i 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative act ivation of the enzyme, because the observed activity changes are relat ed to oxidation of the catalytically important methionine in the activ e site. NMR measurements and amino acid analysis show that oxidation t o methionine sulphoxide of one of five methionines is sufficient to ac tivate alpha-galactosidase. Binding of galactose prevents this. Oxidat ive activation does not lead to conversion of other H2O2-sensitive ami no acid residues, such as histidine, tyrosine, tryptophan and cysteine . The catalytically important cysteine thiol group is quantitatively t itrated after protein oxidative activation. Further oxidation of methi onines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously , a methionine located in the active site of alpha-galactosidase is mo re accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.