Study of the sulfhydryl groups of beta-D-xylosidase from Bacillus pumilus.

Abstract

β‐d‐Xylosidase from Bacillus pumilus contains one intramolecular disulfide bridge and four cysteinyl residues per monomer (Mr 60000). Under denaturing conditions two of these ‐ SH residues react with 5.5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2) with concomitant formation of a second cystine. Under native conditions one cysteinyl group per polypeptide chain reacts with Nbs2 or p‐chloromercuribenzoate (ClHgBzOH) and this results in complete loss of enzymic activity. This inactivation can completely be reversed by reductive treatment (dithiothreitol) of the β‐d‐xylosidase. Kinetic analysis of the inactivation reaction points to a saturation effect, occurring when the concentration of ‐ SH reagent is increased. A reaction model, which includes prior association of the regent with the enzyme, is proposed. Competitive inhibitors protect against inactivation of the enzyme with Nbs2. These experiments prove that two classes of ligands, with two different binding modes, exist. Inactivation with Nbs2 and iodoacetamide as a function of pH reveals the presence of two ionizing groups on the enzyme; their pK values (6.0 and 9.0 respectively) are consistent with those of a protein‐imidazol and protein‐cysteine group. Cumulative evidences obtained with other ‐ SH reagents, such as 5,5′‐dithio bis(2‐nitro‐N‐trimethylbenzylammonium iodide) and iodoacetate, indicate a negatively charged reaction site. Identity with the active site or direct participation of a histidine and/or a cysteinyl group in the enzymic proces can only be suggested.