SUBSTRATE-BINDING TO THE INACTIVE MUTANTS OF STREPTOMYCES SP N174 CHITOSANASE - INDIRECT EVALUATION FROM THE THERMAL UNFOLDING EXPERIMENTS

Oligosaccharide binding to chitosanase from Streptomyces sp, N174 was indirectly evaluated from thermal unfolding experiments of the protein , Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of D-glucosamine oligosaccharides ((GlcN)(n), n=3,4,5, and 6) using the inactive mutant chitosanase in,which the catalytic r esidue, Glu(22), is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A), The midpoint temperature of the unfolding transiti on (T-m) of E22Q was found to be 44.4 degrees C at pH 7.0, However, th e T-m increased upon the addition of (GlcN)(n) by 1.3 degrees C (n = 3 ), 2.5 degrees C (n = 4), 5.2 degrees C (n = 5), or 7.6 degrees C (n = 6), No appreciable change in T-m was observed when (GlcNAc)(6) was ad ded to E22Q, The effect of (GlcN)(n) on the thermal stability was exam ined using the other protein, RNase T1, but the oligosaccharide did no t affect T-m of the protein, Thus,we concluded that the stabilization effect of (GlcN)(n) on the chitosanase results from specific binding o f the oligosaccharides to the substrate binding cleft, When E22D or E2 2A was used instead of E22Q, the increases in T-m induced by (GlcN)(6) binding were 2.7 degrees C for E22D and 4.2 degrees C for E22A. In E2 2D or E22A, interaction with (GlcN)(6) seems to be partly disrupted by a conformational distortion in the catalytic cleft. (C) 1997 Federati on of European Biochemical Societies.