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.

Properties of FNR proteins substituted at each of the five cysteine residues.

FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally-related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine-rich N-terminal extension, which may form part of an iron-binding, redox-sensing domain in FNR. Site-directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non-reducing SDS-PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster-migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N-terminus. FNR(27) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non-essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A-substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene-derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild-type protein may carry a 78 Da substituent which could not be removed with dithiothreitol or beta-mercaptoethanol.

Mass measurement at high molecular weight--new tools for biotechnologists.

It has long been possible to analyse small molecules accurately and reproducibly by mass-spectrometric techniques. Two new techniques extend the application of mass spectrometry to proteins and other biopolymers of high molecular mass. The accuracy, sensitivity and resolving power of these new methods permits the detection of minor, but biologically significant protein modifications.