4 NEW ADENOSINE-DEAMINASE MUTATIONS, ALTERING A ZINC-BINDING HISTIDINE, 2 CONSERVED ALANINES, AND A 5' SPLICE-SITE

Three new missense mutations (H15D, A83D, and A179D) and a new splicin g defect (573 + 1G-->A) in the 5' splice site of intron 5 were among s ix mutant adenosine deaminase (ADA) alleles found in three unrelated p atients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency, When expressed in vitro, th e H15D, A83D, and A179D proteins lacked detectable ADA activity. The s plicing defect caused skipping of exon 5, resulting in premature termi nation of translation and a reduced level of mRNA. H15D is the first n aturally occurring mutation of a residue that coordinates directly wit h the enzyme-associated zinc ion. Molecular modeling based on the atom ic coordinates of murine ADA suggests that the D15 mutation would crea te a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water mole cule postulated to play a role in the catalytic mechanism, A83 and A17 9 are not directly involved in the active site, but are conserved resi dues located respectively in alpha helix 4 and beta strand 4 of the al pha/beta barrel, Replacement of these small hydrophobic Ala residues w ith the charged, more bulky Asp side chain may distort ADA structure a nd affect enzyme stability or folding. (C) 1995 Wiley-Liss, Inc.