On the role of histidine residues in cyclodextrin glycosyltransferase: chemical modification with diethyl pyrocarbonate.

Ethoxyformylation with diethyl pyrocarbonate of approximately 1.5 His residues per molecule of enzyme reduced the cyclising activity of both the alpha-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al and the beta-cyclodextrin glycosyltransferase from Bacillus circulans strain 8 by greater than 90%. Pre-incubation with substrate protected the enzymes from ethoxyformylation. Digestion of starch by the modified enzymes resulted in a delayed formation of cyclodextrins (cyclomalto-oligosaccharides, CDs), but a marked increase in the production of reducing saccharides. Similarly, coupling of alpha CD and maltose and successive disproportionation yielded mainly glucose and malto-oligosaccharides. The results are discussed in the context of the role of conserved His residues for binding of substrate and the transfer reactions.