The biol. function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four b-sheets crosslinking the two lobes, and in a solvent-exposed region below the long b-hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymic functionality of one representative member of the fold family, the human b-secretase, by means of classical mol. dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substrate-catalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estg. the reaction free energy at different representative substrate/enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been obsd. in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topol.-based, energy functional. This anal. reveals a sophisticated mechanism of enzymic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the obsd. fold variations. [on SciFinder (R)]