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 m echanism, structure-function relationships, and more recently for stud ies of pro region-assisted protein folding. We have assigned the backb ones of the enzyme alone, and of its complex with the tetrahedral tran sition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boro Val using doub le-and triple-resonance 3D NMR spectroscopy on uniformly N-15- and C-1 3/N-15-labeled protein. Changes in backbone chemical shifts between th e uncomplexed and inhibited form of the protein are correlated with di stance 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 s econdary structure of the uninhibited enzyme with that of the X-ray st ructure reveals no significant differences. Significant line broadenin g, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), an d in a majority of cases the broadening was reversed upon addition of the inhibitor. Implications and possible mechanisms of this line broad ening are discussed.