Abstract
GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2 This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcat and Km values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.
Highlights
Respectively [5, 6]
The roles of residues in and around the active site of the LodA in catalysis and cysteine tryptophylquinone (CTQ) biogenesis were identified by site-directed mutagenesis [10]
His-466 is of interest because all Group II proteins each have His in this position, which is in the proximity of CTQ in the active site, whereas the Group IA proteins all have a Cys at the corresponding position
Summary
The concentration of the glycine was varied in the presence of 100% O2-saturated (1150 M) buffer. The concentration of O2 was varied in the presence of a saturating concentration of glycine (5 mM) In this case, the data fit well to the Michaelis-Menten equation (Fig. 3C), and no improvement of the fit was obtained by using the Hill equation (Fig. 3D). Because the apparent Km for O2 is much larger than concentration in a 100% O2-saturated solution, the perceived saturation kinetics in Fig. 3C is largely due to the single data point at 1150 M, yielding relatively large errors in kcat/Km that are determined from the fitted parameters. The kcat obtained when varying O2 was 93 sϪ1 as compared with 39 sϪ1 when varying glycine The reason for this is that the fixed concentration of 100% O2-saturated buffer that was used, which is the highest that can be achieved, was not a saturating concentration of substrate with respect to the enzyme (i.e. it was not much greater than the Km for O2).
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