On the origin of the broad-band selectivity of bovine-lens-leucine-aminopeptidase

Abstract

Detailed DFT (B3LYP) calculations on the mode of action of bovine lens leucine aminopeptidase (blLAP), a binuclear zinc enzyme that hydrolyzes the N-terminal residue of a peptide chain, provide first explanations for the unusual coordination sphere at Zn(1) as well as the presence of a second water channel. The unusual architecture of the active site promotes the spontaneous generation of a water sluice that continually regenerates a series of species capable of acting as active nucleophiles. We now demonstrate that this sluice mechanism is coupled with substrate docking. Both possibilities lead to a complicated hypersurface in which a multitude of stable intermediates and possible nucleophilic species are generated which are in thermodynamic equilibrium with each other. The hypersurface is characterized by very low barriers for interconversion between the intermediates – all of which possess similar stabilities - and thus provides a first explanation for the broad-band activity of blLAP which is capable of hydrolyzing all amino acids except for proline and arginine. Most steric and electronic constraints in the substrate can be overcome by simply selecting a different pathway on the hypersurface. First calculations indicate that a “classical” twostep hydrolysis over a gem-diol/diolate intermediate has a prohibitively high barrier and is thus a probable dead-end on the hypersurface. Alternatively, a non-classical mechanism in which the nucleophilic attack on the carbonyl bond as well as the C-N bond cleavage occurs simultaneously over a chair-like ring transition structure is postulated.