Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

Site-directed mutagenesis has been used to characterize the functions of ke y amino acid residues in the catalytic site of the 'nudix' hydrolase, (asym metrical) diadenosine 5 ' ,5 ' ' ' -P-1,P-4-tetraphosphate (Ap(4)A) hydrola se (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix mot if, Gly-(Xaa)(5)-Glu-(Xaa)(7)-Arg-Glu-Uaa-Xaa-(Glu)(2)-Xaa-Gly (where Xaa r epresents unspecified amino acids and Uaa represents the bulky aliphatic am ino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues impor tant for catalysis from elsewhere in the molecule, were mutated and the exp ressed proteins characterized. The results reveal a high degree of function al conservation between lupin asymmetric Ap(4)A hydrolase and the 8-oxo-dGT P hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP h ydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674 ] were shown to contribute to catalysis to similar extents in the lupin enz yme. Mutations E55Q, E59Q and E125Q all reduced k(cat) markedly, whereas mu tations R54Q, E58Q and E122Q had smaller effects. None of the mutations pro duced a substantial change in the K-m for Ap(4)A but several extensively mo dified the pH-dependence and fluoride-sensitivities of the hydrolase. It wa s concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hy drolytic mechanism in both of these members of the nudix family of hydrolas es.