STRUCTURE-FUNCTION RELATIONSHIP OF XYLANASE - FLUOROMETRIC ANALYSIS OF THE TRYPTOPHAN ENVIRONMENT

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 10 41, 172-177]. The work described here aims at: (i) deducing the struct ure-function relationship for the tryptophan residue involved at the a ctive site (a) by correlating the effect of N-bromosuccinimide (NBS) o n the fluorescence and activity, and (b) by assessing the ability of x ylan to protect against decrease in fluorescence versus activity of NB S-treated enzyme; and (ii) probing into the environment of the tryptop han residues by studying the quenching of their fluorescence by variou s solute quenchers in the presence and absence of guanidine hydrochlor ide (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tr yptophan at the substrate-binding site of the xylanase. The xylanase e xhibited a rather low fluorescence emission maximum at 310 nm. There w as no shift in the emission maximum on treatment of the enzyme with Gd n.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested t he occurrence of both collisional as well as static quenching processe s. The enzyme retained full activity as well as the characteristic emi ssion maximum at 310 nm in the presence of acrylamide (100 mM), indica ting that quenching of fluorescence by acrylamide is a physical proces s. Acrylamide was more efficient as a quencher than CsCl or KBr. Treat ment of the enzyme with Gdn.HCl resulted in an increase in accessibili ty of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K-ST) of the solute quenchers. T he analysis of K-ST and V values of KBr and CsCl suggests that the ove rall tryptophan microenvironment in the xylanase from Chainin is sligh tly electronegative.