Allosteric activation of the B. subtilis aminopeptidase BSAP: towards a “perfect” enzyme

Aminopeptidases catalyze the cleavage of a 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 was to explore the structure-function relationship of the bacterial metallo-aminopeptidase from Bacillus subtilis (BSAP). The detailed three-dimensional structure of BSAP was determined by X-ray crystallography at 1.7Å resolution. The 46kDa enzyme consists of two main domains; a TIM barrel catalytic domain (AA 32-100, 226-455) containing two Zn molecules and a protease-associated (PA) domain (AA 101-225). Interestingly, the 23-AA C-terminus tail appeared unstructured in the crystal structure, with no clear electron density for most of it. However, Glu452 at the end of the tail interacted strongly with one of the Zn ions inside the active site, potentially interfering with substrate binding. To study the exact roles of the carboxy-terminus tail of BSAP, we prepared a series of truncated forms of the enzyme, including deletions and key amino acid replacements. The removal of the whole C-terminus tail did not affect the activity towards the chromogenic substrate p-nitroanilide-Lys and several peptides examined, suggesting that Glu452 is not inhibiting activity. Unexpectedly, however, a single replacement Glu452Asn resulted in a 3-orders of magnitude increase of kcat without affecting significantly KM, providing a 3000-fold improvement in the specificity constant kcat/KM. The δ-amine group of Asn452 appears to stabilize the transition state, thus reducing the free activation energy by up to 6.6 Kcal/mole. Kinetic measurements at different viscosities indicated the rate of hydrolysis is under diffusional-control, consistent with the observed value of the specificity constant, 9.4×108 M-1s-1 against the hexa-peptide KAAAAW. Our results demonstrate that BASP can be activated allosterically making it a “perfect enzyme”.