Mutation of residues in the coenzyme binding pocket of Dopa decarboxylase - Effects on catalytic properties

Residues D271, H192, H302 and N300 of l-3,4-dihydroxyphenylalanine decarbox ylase (DDC), a homodimeric pyridoxal 5'-phosphate (PLP) enzyme, were mutate d in order to acquire information on the catalytic mechanism. These residue s are potential participants in catalysis because they belong to the common PLP-binding structural motif of group I, II and III decarboxylases and oth er PLP enzymes, and because they are among the putative active-site residue s of structural modelled rat liver DDC. The spectroscopic features of the D 271E, H192Q, H302Q and N300A mutants as well as their dissociation constant s for PLP suggest that substitution of each of these residues causes altera tion of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This su pports, but does not prove, the possibility that these residues are located in the coenzyme-binding cleft. Interestingly, mutation of each residue gen erates an oxidative decarboxylase activity towards l-3,4-dihydroxyphenylala nine (l-Dopa), not inherent in the wild-type in aerobiosis, and reduces the nonoxidative decarboxylase activity of l-Dopa from 3- to 390-fold. The par tition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 x 10(-4) for N300A mutant to 946 x 10(-4) for H302Q mutant. Unlike wil d-type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O-2. In addition, all four mutants exh ibit an extremely low level of the oxidative deaminase activity towards ser otonin with respect to wild-type. All these findings demonstrate that altho ugh D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship amon g the integrity of the PLP cleft, the productive binding of O-2 and the tra nsition to a closed conformational state of DDC is discussed.