Site-directed mutagenesis and molecular modelling studies show the role of asp82 and cysteines in rat acylase 1, a member of the m20 family

Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis expts. led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a crit. role in the enzymic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, resp., along with a loss of zinc coordination. Mol. modeling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addn., none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure.