Kinetic Studies on β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE)

Abstract

The steady-state kinetic mechanism of ␤-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 ratelimiting iso-step occurs after formation of a tetrahedral intermediate. A moderately short and strong hydrogen bond (at ␦ 13.0 ppm and 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 ␤-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.Extracellular amyloid deposits in brain, a characteristic feature of Alzheimer's disease, is a result of proteolytic cleavage of membrane-bound amyloid precursor protein by two enzymes, ␤-secretase and ␥-secretase. The second cleavage activity (␥-secretase) is strongly associated with the presenilin multisubunit complexes (1), whereas ␤-secretase (BACE) 1 has been identified as a novel transmembrane aspartyl protease (2-4).Although aspartyl proteases have been studied for more than 4 decades, new aspects of catalysis and inhibition continue to emerge. A substantial number of these enzymes have been identified as useful targets for chemotherapeutic intervention in human diseases (5-8), yet there has been limited success in identifying clinically relevant inhibitors; hence, it is important to explo...

Crystal Structure of an Active Form of BACE1, an Enzyme Responsible for Amyloid β Protein Production

“…However, such H bond interactions may not exist or may decrease in the diprotonated form in acidic conditions, resulting in the destabilization of Wat1. It has been proposed that a transition between the hydrated and dehydrated forms is also present in the apo form, and Wat1 might be exchanged with other water molecules, since some inhibitors bind only to the dehydrated form of human immunodeficiency virus protease (25), and the same mechanism probably operates in BACE1 (32). Thus, it seems likely that there is a pH-dependent equilibration between the hydrated form and the dehydrated form, and the dehydrated form is expected to be predominant at a pH level less than 4.0.…”

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

“…Methodologically, fluorogenic substrates as well as matrix-assisted laser desorption ionization time-of-flight and HPLC were applied to investigate cleavage. From these analyses K m values from 4.5 M to 1 mM were estimated (37,41,47,60,63). To our knowledge, kinetic parameters for BACE-FL have only been published by Kopcho et al (63) so far.…”

“…Moreover, a participation of native BACE in a larger complex could alter its enzymatic properties (39,47). Because BACE is the rate-limiting enzyme of A␤ production (4, 31), a detailed understanding of its molecular architecture and function is of great importance.…”

NMR Study of the Inhibition of Pepsin by Glyoxal Inhibitors: Mechanism of Tetrahedral Intermediate Stabilization by the Aspartyl Proteases

“…nificantly different from those of earlier studies on -secretases (16) and endathiapepsin (17,18). We discuss the role of hydrogen bonding in stabilizing gem-diols formed during catalysis and on binding inhibitors.…”