The chemical modification of tryptophan residues of alpha-mannosidase from Phaseolus vulgaris.

Reaction of alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from Phaseolus vulgaris with N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide- resulted in loss of enzyme activity. Spectral absorption and fluorescence studies, as well as amino acid analysis, suggested that only tryptophan residues had been modified. No change in conformation could be detected by density gradient ultracentrifugation or circular dichroism of alpha-mannosidase modified by N-bromosuccinimide to virtually zero enzyme activity. The inhibition was partly offset by the substrate analogue alpha-methyl-D-mannoside and the competitive inhibitor mannono-1,4-lactone. Concomitantly, two tryptophan residues fewer were oxidized per molecule. After modification V was reduced, while Km seemed unchanged. Further, there was found evidence for the enzyme having a secondary structure dominated by beta-pleated sheets.