Kinetic studies on beta-site amyloid precursor protein-cleaving enzyme (BACE). Confirmation of an iso mechanism

The steady-state kinetic mechanism of beta-amyloid precursor protein-cleaving enzyme (BACE)-catalyzed proteolytic cleavage was evaluated using product and statine- (Stat(V)) or hydroxyethylene-containing (OM99-2) peptide inhibition data, solvent kinetic isotope effects, and proton NMR spectroscopy. The noncompetitive inhibition pattern observed for both cleavage products, together with the independence of Stat(V) inhibition on substrate concentration, suggests a uni-bi-iso kinetic mechanism. According to this mechanism, the enzyme undergoes multiple conformation changes during the catalytic cycle. If any of these steps are rate-limiting to turnover, an enzyme form preceding the rate-limiting conformational change should accumulate. An insignificant solvent kinetic isotope effect (SKIE) on k(cat)/K(m), a large inverse solvent kinetic isotope effect on k(cat), and the absence of any SKIE on the inhibition onset by Stat(V) during catalysis together indicate that the rate-limiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at delta 13.0 ppm and phi of 0.6) has been observed by NMR spectroscopy in the enzyme-hydroxyethylene peptide (OM99-2) complex that presumably mimics the tetrahedral intermediate of catalysis. Collapse of this intermediate, involving multiple steps and interconversion of enzyme forms, has been suggested to impose a rate limitation, which is manifested in a significant SKIE on k(cat). Multiple enzyme forms and their distribution during catalysis were evaluated by measuring the SKIE on the noncompetitive (mixed) inhibition constants for the C-terminal reaction product. Large, normal SKIE values were observed for these inhibition constants, suggesting that both kinetic and thermodynamic components contribute to the K(ii) and K(is) expressions, as has been suggested for other iso-mechanism featuring enzymes. We propose that a conformational change related to the reprotonation of aspartates during or after the bond-breaking event is the rate-limiting segment in the catalytic reaction of beta-amyloid precursor protein-cleaving enzyme, and ligands binding to other than the ground-state forms of the enzyme might provide inhibitors of greater pharmacological relevance.