THE SOLUTION STRUCTURE OF SERINE-PROTEASE PB92 FROM BACILLUS-ALCALOPHILUS PRESENTS A RIGID FOLD WITH A FLEXIBLE SUBSTRATE-BINDING SITE

Background: Research on high-alkaline proteases, such as serine protea se PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensi vely studied. These studies have included exhaustive protein engineeri ng investigations and X-ray crystallography, in order to provide insig ht into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crys tal structures, due to crystal contacts. In addition, the structural v ariability in the substrate-binding region of subtilisins is often att ributed to flexibility. It was hoped that the solution structure of th is enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of th ese enzymes. Results: The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using di stance and dihedral angle constraints derived from triple-resonance NM R data. The solution structure is represented by a family of 18 confor mers which overlay onto the average structure with backbone and all-he avy-atom root mean square deviations (for the main body of the molecul e) of 0.88 and 1.21 Angstrom, respectively. The family of structures c ontains a number of regions of relatively high conformational heteroge neity, including various segments that are involved in the formation o f the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and meas urements of amide proton exchange rates. Conclusions: The solution str ucture of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Am ong the limited number of residues involved in significant internal mo bility are those of two pockets, termed S1 and S4, within the substrat e-binding site. The presence of flexibility within the binding site su pports the proposed induced fit mechanism of substrate binding.