Residues D271, H192, H302 and N300 of l-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5 H -phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP-binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active-site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration 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 supports, but does not prove, the possibility that these residues are located in the coenzyme-binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards l-3,4-dihydroxyphenylalanine (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 partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 Â 10 24 for N300A mutant to 946 Â 10 24 for H302Q mutant. Unlike wild-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 exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild-type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O 2 and the transition to a closed conformational state of DDC is discussed.
Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kc,, values of about 50% and 15%, respectively, at pH 6.8, while the K, values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (LJ-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-1 1 ]-+Ala mutant displays a reduced affinity for pyridoxal 5"phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pK, and pKb for the groups involved in catalysis were determined for the wild-type and the C l l l A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 -+ 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-1 1 1 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur JBiochern 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.Keywords: active site; decarboxylase; pyridoxal 5"phosphate; site-directed mutagenesis The versatility of pyridoxal 5"phosphate (PLP) as a co-factor is demonstrated by the wide variety of reactions that enzymes dependent upon it catalyze. Although detailed catalytic mechanisms and information correlating structure and function have been reported for many PLP dependent enzymes, such information is conspicuously lacking for PLP dependent decarboxylases.Even though aromatic amino acid decarboxylases have been cloned from a number of mammalian (Ichinose et al., 1989;Tanaka et al., 1989;Kang & Joh, 1990;Taketoshi et al., 1990), insect (Eveleth et al., 1986, and plant sources (DeLuca et al., 1989;Kawalleck et al., 1993; Facchini & DeLuca, 1994)
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