The Glu452Asn replacement in the B. subtilis aminopeptidase (BSAP) increases the specificity constant by a 1000-fold

Aminopeptidases catalyze the cleavage of single amino acid from the amino terminus of peptides and proteins and are involved in many crucial biological processes. The goal of the current study is to explore the structure-function relationship of the bacterial metallo-aminopeptidase from Bacillus subtilis (BSAP). The detailed three-dimensional structure of BSAP has recently been determined by X-ray crystallography, at 1.77Å resolution. The enzyme consists of two main domains; a TIM barrel catalytic domain with two Zn molecules and a protease-associated (PA) domain. Based on the structure, the C-terminal tail (23 AA) appears to interact with the PA-domain near the active site and potentially blocking it. The main interactions are made by residues Asp155 and Tyr158 in the PA- domain. In addition, Glu452 appears to coordinate tightly with one of the two Zn ions in the active site, thereby potentially interfering with substrate binding. To study the exact roles of the PA domain and the carboxy-tail of BSAP, we prepared a series of truncated forms of the enzyme, including deletions and key amino acid replacements. Surprisingly, the Asp155Ala, Tyr158Phe, and Glu452Ala replacements and the complete removal of the C-terminus tail, did not affect significantly the activity towards the chromogenic substrates p-nitroanilide-Arg and -Lys. Unexpectedly, however, the Glu452Asn replacement resulted in a 1000-fold improvement in the specificity constant k_cat/K_M. The kcat and K_M were 9.5 sec^-1, 1.2 mM and 9.7x102 sec^-1, 1.0x10^-1 mM for BSAP and BSAP (Glu452Asn), respectively. This increase in activity could not be explained by change in enzyme flexibility since the melting temperature of the Glu452Asn mutant increased by ~5°C. Thus, the δ-amine group of Asn452 appears to stabilize the transition state in a yet unknown mechanism.