Engineered Bacillus lentus subtilisins having altered flexibility.

The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and N76D/N87S/S103A/V104I (DSAI), were determined by X-ray crystallography. In addition to identifying changes in atomic position we report a method that identifies protein segments having altered flexibility. The method utilizes a statistical analysis of variance to delineate main-chain temperature factors that represent significant departures from the overall variance between equivalent regions seen throughout the structure. This method reveals changes in main-chain mobility in both variants. Residues 125-127 have increased mobility in the RSYSA variant while residues 100-104 have decreased mobility in the DSAI variant. These segments are located at the substrate-binding site and changes in their mobility are believed to relate to the observed changes in proteolytic activity. The effect of altered crystal lattice contacts on segment flexibility becomes apparent when identical variants, determined in two crystal forms, are compared with the native enzyme.

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin.

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.

Active-site titration of serine proteases using a fluoride ion selective electrode and sulfonyl fluoride inhibitors.

We report a general procedure for the determination of active enzyme concentrations for serine proteases. The method relies on the measurement of fluoride ion released from sulfonyl fluorides upon reaction with the active-site serine using an ion selective electrode. The results have been independently confirmed by amino acid analyses of subtilisins and by spectrofluorometric and spectrophotometric titrations. The minimal enzyme concentration detectable is 1-10 microM protease. The method is insensitive to color and turbidity of the sample and is therefore useful for measuring protease concentration in broth solutions. The active enzyme concentration of subtilisin BPN' from Bacillus amyloliquefaciens determined by titration with phenylmethylsulfonyl fluoride is 25% higher than the concentration determined using the spectrophotometric burst titrant N-trans-cinnamoylimidazole. Analysis of the pre-steady-state burst amplitude and kinetics suggests that the extinction coefficient for the cinnamoyl acyl-enzyme is larger than previously measured and a significant fraction of the enzyme is present as an unproductive ES2 complex. The molar extinction coefficient at 280 nm for subtilisin BPN' is 26.5 mM-1 cm-1 and for subtilisin from Bacillus lentus is 22.5 mM-1 cm-1.

Packing selection of Bacillus lentus subtilisin and a site-specific variant.

The crystallization of a variant of Bacillus lentus subtilisin and the native enzyme was achieved using identical conditions. The variant B. lentus was found to grow in two crystal forms, form 1 and form 2, whereas the native B. lentus subtilisin enzyme crystallized in only one, form 1. Form 2 crystals, once obtained, were found to grow much more rapidly than form 1 crystals. The lattice contacts and structural changes giving both crystal forms have been examined. The results show that crystal form 2 has a more complex network of interactions. There is also a small surface conformational change in the form 2 structure relative to the native and variant form 1 crystals and at least two solvent molecules bound to the enzyme in crystal form 1 are displaced in crystal form 2. In addition, a site specific substitution in the variant at position 27 induces a 'short' lattice contact which does not exist in the native B. lentus or the form 2 variant B. lentus. These results suggest that in some circumstances engineered variants could be designed to crystallize more rapidly than the native enzyme.