Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.
alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase 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; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (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 more 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.
Kachurin
A M
AM
Department of Molecular and Radiation Biophysics, St. Petersburg Nuclear Physics Institute, Gatchina, Russia.
Golubev
A M
AM
Geisow
M M
MM
Veselkina
O S
OS
Isaeva-Ivanova
L S
LS
Neustroev
K N
KN
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003543:Cysteamine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005697:Galactosides / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008715:Methionine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D005697:Galactosides; D016877:Oxidants; D013439:Sulfhydryl Reagents; D003543:Cysteamine; D008715:Methionine; D006861:Hydrogen Peroxide; D000519:alpha-Galactosidase; C013111:methionine sulfoxide