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Bacillus circulans xylanase was used as a model system to study the effect of acid/base catalyst and its spatial positioning on the functional activity of glycosidase. The results of kinetic studies involving B. circulans xylanase bearing Glu172Asp mutation confirmed that the hydrolysis of xylan is greatly influenced by the distance between the two catalytic carboxylic acids of the enzyme. The enzymatic activity was completely abrogated with the removal of the carboxyl group although the absence of such acid/base catalyst can be compensated for by the addition of an anionic nucleophile in the reaction system. The results thus confirm the role of Glu172 as an acid/base catalyst and the importance of carboxyl groups at such position.

The position of the two carboxyl groups in Bacillus circulans xylanase has an important effect on its catalytic action. Mutagenesis of the catalytic nucleophile Glu78 helps to change the distance between the two groups. The rate of glycosylation reduces drastically due to an increase in the separation. Catalytic activity reduces to a lesser degree when there is a decrease in the separation. The rate differences are not reflected in the extent of bond cleavage or proton donation at the transition state. Removal of the carboxyl group (Glu78Cys) stops catalytic activity.

The thiol exchange reaction is investigated using rapid flow-quench kinetic studies of the reaction between thiols and ESSG, the form of the enzyme in which glutathione is covalently attached to Cys13. The results suggest that the second glutathione binds very weakly and that its pK(super a) is not significantly perturbed from that observed in solution.

An enzyme that catalyzes an iron-dependent reaction of 2,6-dichlorohydroquinone with O2 is purified from Sphingomonas chlorophenolica strain ATCC 39723, which is capable of complete mineralization of pentachlorophenol. A sequence analysis reveals that the enzyme is an O2-dependent ring cleavage dioxygenase. Moreover, the enzyme is encoded by the pcpA gene and appears to belong to a rare class of extradiol dioxygenases.