Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to alpha-lytic protease

alpha-lytic protease, a bacterial serine protease of 198 amino acids (19 800 Da), has been used as a model system for studies of catalytic mechanism, structure-function relationships, and more recently for studies of pro region-assisted protein folding. We have assigned the backbones of the enzyme alone, and of its complex with the tetrahedral transition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boro Val using double-and triple-resonance 3D NMR spectroscopy on uniformly N-15- and C-13/N-15-labeled protein. Changes in backbone chemical shifts between the uncomplexed and inhibited form of the protein are correlated with distance from the inhibitor, the displacement of backbone nitrogens, and change in hydrogen bond strength upon inhibitor binding (derived from previously solved crystal structures). A comparison of the solution secondary structure of the uninhibited enzyme with that of the X-ray structure reveals no significant differences. Significant line broadening, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), and in a majority of cases the broadening was reversed upon addition of the inhibitor. Implications and possible mechanisms of this line broadening are discussed.