Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins

High-alkaline serine proteases have been successfully applied as protein de grading components of detergent formulations and are subject to extensive p rotein engineering efforts to improve their stability and performance. Dyna mics has been suggested to play an important role in determining enzyme act ivity and specificity and it is therefore of interest to establish how loca l changes in internal mobility affect protein stability, specificity and pe rformance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, th at has improved washing performance. T-1, T-2 and heteronuclear NOE measure ments of the N-15 nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 an d S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the cataly tic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonst rate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop re gions involved in substrate binding become more structured in the engineere d variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been est ablished by X-ray crystallograpic methods, as shown in the accompanying pap er. (C) 1999 Academic Press.