Mutational analysis of a proposed glycine binding site in PEP carboxylase

PEP carboxylase from maize is a tetrameric enzyme that is allosterically activated by glycine. G937 and K927 are part of a pocket at the subunit interface that we propose to be the glycine binding site. This pocket has several positive charges at its surface, and flanking this pocket is a 7‐residue mobile loop (residues 929‐35). The mutant K927Q shows reduced activation, suggesting that positive charge in this location is important for glycine binding. Similarly, G937D is significantly desensitized to glycine. This latter mutation mimics a structural feature of PEP carboxylase from dicot plants and may explain why dicot enzymes are unresponsive to glycine. Removal of positive charge (K934Q) from the nearby mobile loop weakened glycine binding, while the removal of negative charge (E932Q) from the loop enhanced the enzyme’s affinity for glycine. Mutation of the nearby lysine 940 (K940Q) also partially reduced the response to glycine, but other nearby mutations, including N933G and C335S, did not significantly reduce the enzyme’s affinity to the activator glycine, although interestingly N933G did show inhibition at very high levels of glycine. These results support the hypothesis that glycine binds at this pocket, and that the adjacent mobile loop contributes to the binding of this activator. LG was supported through NIH MBRS RISE award R25 GM61331 and JH was supported by a MARC Traineeship through grant T34 GM 08228.