MUTATION OF CYSTEINE-111 IN DOPA DECARBOXYLASE LEADS TO ACTIVE-SITE PERTURBATION

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzy me, the resultant C111A and C111S mutant enzymes exhibit k(cat) values of about 50% and 15%, respectively, at pH 6.8, while the K-m values r emain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) a nd L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of th e 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are si milar to those of the wild type. With respect to the wild type, the Cy s-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosph ate (PLP), slower kinetics of reconstitution to holoenzyme, a decrease d ability to anchor the external aldimine formed between D-Dopa and th e bound co-enzyme, and a decreased efficiency of energy transfer betwe en tryptophan residue(s) and reduced PLP. Values of pK(a) and pK(b) fo r the groups involved in catalysis were determined for the wild-type a nd the C111A mutant enzymes. The mutant showed a decrease in both pK v alues by about 1 pH unit, resulting in a shift of the pH of the maximu m velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximu m velocity is mirrored by a similar shift in the spectrophotometricall y determined pK value of the 420 --> 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys- 111 is catalytically nonessential and provide strong support for previ ous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattomi C. 1991. fur J Bio chem 201:393-397). Moreover, our findings provide evidence that Cys-Il l has a structural role in PLP binding and suggest that this residue i s required for maintenance of proper active-site conformation.