X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state.

We have studied the structures of adducts formed between subtilisin BPN' and both benzeneboronic acid and 2-phenylethaneboronic acid by x-ray diffraction techniques. Electron density and difference maps at 2.5 A resolution were computed with phases calculated from a partially refined structure of the native enzyme (R = 0.23 at 2.0 A). Both adducts contain a covalent bond between Ogamma of the catalytic Ser-221 and the inhibitor boron atom. The boron atom is coordinated tetrahedrally, with one of the two additional boronic acid oxygen atoms lying in the "oxyanion hole" and the other at the leaving group site identified in previous studies (ROBERTUS, J.D., Kraut, J. ALDEN, R.A., and BIRKTOFT, J.J. (1972) Biochemistry 11, 4293-4303). Moreover, the previously postulated structure of the tetrahedral intermediate for substrate hydrolysis is isosteric with these boronic acid adducts, which can therefore be considered good models for the transition state complex (KOEHLER, K.K., and LIENHARD, G.E. (1972) Biochemistry 10, 2477-2483). These observations further support the suggestion that an important contribution to stabilization of this transition state complex, relative to both the Michaelis complex and the acyl intermediate, occurs as a consequence of hydrogen bond donation to the substrate carbonyl oxygen atom from the side chain amido group of Asn-155 and from the backbone amido group of Ser-221.