AdoMetDC belongs to a class of pyruvoyl-dependent enzymes that includes aspar-tate decarboxylase, phosphatidylserine decarboxylase, and histidine decarboxylase (41). The catalytic activities of these enzymes rely on a covalently bound pyruvoyl group rather than the pyridoxal-5'-phosphate cofactor more commonly employed by amino acid decarboxylases. Pyruvoyl enzymes are synthesized as an inactive proenzyme that undergoes an apparently autocatalytic internal cleavage reaction to yield two subunits (in the case of AdoMetDC, a larger C-terminal-derived a subunit and a smaller N-ter-minal-derived P subunit). In mammalian AdoMetDC, the cleavage site is located between Glu-67 and Ser-68 within the conserved motif YVLSESS, and the pyruvoyl group is formed by conversion of the resulting serine residue at the amino terminus of the a subunit (42).
The chemical mechanism for formation of the AdoMetDC pyruvoyl involves nucle-ophilic attack by the hydroxyl group of Ser-68 at the carbonyl carbon atom of Glu-67, resulting in the formation of an oxyoxazolidine intermediate. This intermediate undergoes an N^O acyl rearrangement to form an ester intermediate, which in turn undergoes a P elimination reaction to form the a subunit with a dehydroalanine residue at its N-terminus, and the P subunit. The dehydroalanine is then converted to pyruvoyl with the loss of ammonia via the formation of imine and carbinoalamine intermediates (43)
The crystal structure of wild-type AdoMetDC in its processed form revealed that residue Glu-67 remains close to the pyruvate residue, indicating that the protein does not undergo a major conformational change after cleavage, and hence conserved residues within the active site pocket are also candidates for a role in processing (4). Three such residues are Cys-82, Ser-229, and His-243, and mutation of these amino acids resulted in impaired proenzyme processing. Mutant C82A processed approximately 10 times more slowly than the wild-type enzyme (44). Mutant S229A did not process, S229C processed at a much reduced rate, and S229T processed normally, suggesting that the hydroxyl group of Ser-229 is critical to the reaction (45). Mutant H243A cleaved very slowly, with the majority of the resulting a subunit having an N-terminal serine rather than a pyruvoyl, but cleavage was accelerated by addition of hydroxylamine (45). This latter result is indicative of cleavage occurring via hydrolysis of the ester intermediate rather than P elimination, and solving of the crystal structure of the H243A mutant revealed that the protein is indeed trapped in the ester form, confirming that it is the P elimination step that is prevented in this mutant (46). Based on these observations and modeling of the oxyoxazolidine intermediate from the crystal structure of the nonprocessing S68A mutant, Tolbert et al. (43) proposed a processing mechanism in which the formation of the oxyoxazolidine intermediate is promoted by
a hydrogen bond from Cys-82 and then stabilized by a hydrogen bond from Ser-229. Donation of a proton by His-243 to the nitrogen atom of the oxyoxazolidine intermediate then leads to formation of the ester intermediate. Because the H243A mutant enzyme is able to form the ester intermediate (46), Tolbert et al. (43) postulated that a water molecule may act as a substitute proton donor in this mutant. The suggested mechanism for the next step in the processing reaction is abstraction of the Ha proton of Ser-68 by the His-243 side chain, resulting in cleavage of the peptide chain and formation of the dehydroalanine residue (46).
The rate of the mammalian AdoMetDC processing reaction is accelerated by the addition of putrescine (47), and the four acidic residues Glu-11, Asp-174, Glu-178, and Glu-256 are necessary for this stimulation (48). A single putrescine molecule was bound to each monomer of the H243A mutant, within a cluster of buried negatively charged residues between the two P sheets, about 15-20 A from the active site (46). The presence of a single bound putrescine molecule was unexpected for two reasons. First, putrescine had not been added during the crystallization process, leaving the Escherichia coli protein expression system as the most likely source. Second, kinetics data from experiments with putrescine and two substituted analogs had strongly suggested two or more binding sites for putrescine (8). The single putrescine molecule observed in H243A forms direct interactions with the side chains of Glu15, Asp174, and Thr176, and indirect water-mediated interactions with side chains of Glu15, Ser113, Glu178, and Glu256. Furthermore, this putrescine-binding site is linked to the active site by a network of hydrogen bonds, involving residues Glu-11, Lys-80, and His-243 (46). These observations led Ekstrom et al. (46) to postulate that putrescine binding serves to balance the negative charges of the binding site, thereby repositioning critical active site residues, either by causing a shift in the relative orientations of the P sheets, or by charge transmission via the hydrogen bond network, or both. However, the structure of the proenzyme (i.e., the S68A nonprocessing mutant) and the modeled structure of the oxyoxazolidine intermediate demonstrate that neither Glu-11 nor Lys-80 interact with Cys-82 or Ser-229, leading to the conclusion that putrescine influences processing mainly via an effect on His-243 (43).
Unlike the mammalian enzyme, plant AdoMetDCs undergo very rapid autoprocess-ing and do not require putrescine for an optimal processing rate (48). The crystal structure of the potato AdoMetDC provides an explanation for this difference (49). Two amino acid changes in the buried putrescine-binding site, Leu-13/Arg-18 and Phe-111/Arg114, indicate that two arginine residues could substitute for putrescine in balancing the negative charge of the site. Unlike putrescine, which binds reversibly to the mammalian enzyme, these arginine residues are always present, so the enzyme will always be in the activated state, thus explaining the apparent rapid autoprocessing of plant AdoMetDCs. The potato AdoMetDC structure also revealed that there is a very similar network of hydrogen bonds between the charged site and the active site as seen in the human AdoMetDC, supporting the proposed role of this network in transmission of the putrescine/arginine effect. Interestingly, the potato AdoMetDC does not form a dimer, but rather a monomer (aP). This is probably owing to substitutions of several residues that, in the human AdoMetDC, form interactions across the dimer interface;
but it may also have significance in regulation of the respective enzymes because disruption of the human dimer interface would probably make the putrescine-binding site more exposed (49).
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