Conformation and polarity of the active site of xylanase I from Thermomonospora sp as deduced by fluorescent chemoaffinity labeling - Site and significance of a histidine residue

A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascer tain the conformational flexibility and polarity at the active site of xyla nase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed th at complete inactivation occurred due to the binding of one molecule of OPT A to the active site of Xyl I. The formation of a single fluorescent isoind ole derivative corroborated these findings. OPTA has been known to form a f luorescent isoindole derivative by crosslinking the proximal thiol and amin o groups of cysteine and lysine. The involvement of cysteine in the formati on of a Xyl I-isoindole derivative has been negated by fluorometric and che mical modification studies on Xyl I with group-specific reagents and by ami no-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the cataly tic site of Xyl I. Modification of histidine and lysine residues by diethyl pyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolish ed the ability of the enzyme to form an isoindole derivative with OPTA, ind icating that histidine and lysine participate in the formation of the isoin dole complex. A mechanism for the reaction of OPTA with histidine and lysin e residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site o f Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fl uorescence emission maxima of a model compound (isoindole adduct) in solven ts of different polarity were compared with the fluorescence behaviour of t he Xyl I-isoindole derivative, leading to the conclusion that the active si te is located in a microenvironment of low polarity.