STRUCTURAL AND FUNCTIONAL-ROLE OF TRYPTOPHAN IN XYLANASE FROM AN EXTREMOPHILIC BACILLUS - ASSESSMENT OF THE ACTIVE-SITE

Microenvironment and conformation of the active site of xylanase from an extremophilic Bacillus was deciphered for the first time using fluo rescence spectroscopy. NBS modified enzyme showed complete inactivatio n and the kinetic analysis implicated the presence of an essential try ptophan at the active site of xylanase, Xylan (0.5%) protected the enz yme completely from inactivation with NBS, whereas it afforded 35% pro tection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site. Quenching stud ies revealed that acrylamide was more efficient than KI and CsCl as in dicated by the higher Stern-Volmer quenching constants (Ksv), The ster ic factor represented by the percentage accessibility of the tryptopha n residues of XylII was higher with the positively charged Cs+ (80) th an with the negatively charged I- (10), suggesting that the tryptophan residues are located in a relatively electronegative environment. In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with in creased accessibility of the fluorophore to the quenchers. The proximi ty of the essential carboxyl groups with a high pKa value of 6.9 [Chau thaiwale and Rao (1994) Biochim. Biophys. Acta] probably contributes t o the electronegative environment of the tryptophan residue. Our resul ts on sequence analysis of the gene encoding for XylII (Accession Numb er U83602 in the GenBank database) have shown that Trp 61 is highly co nserved and may play a role in the structure-function relationship of the enzyme. (C) 1998 Academic Press.