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Probing the Active Site of Thermotoga Maritima ADPGlucose Pyrophosphorylase

The T. maritima ADP‐glucose pyrophosphorylase (ADPG PPase) is comprised of both glgC and glgD subunits which form a heterotetramer. While glgD displays negligible activity alone, the low activity glgC showed ~20‐fold stimulation when combined with glgD and also responds to the activator FBP. Amino acid alignment and modeling studies indicated that glgC D126 may be a catalytic residue, so this position was probed by site‐directed mutagenesis. D126A was generated and the His tagged protein purified via a heat step and nickel affinity chromoatography. The D126A enzyme displayed a ~1400 fold decrease in activity compared to the native enzyme while the S0.5 for ATP only increased ~2‐fold. Native PAGE revealed that the D126A protein successfully formed a complex with WT glgD which was also activated by FBP. The glgC D126 is an important residue for catalysis indicating that the enzyme active site is similar to other bacterial ADPG PPases. Additional analyses of D mutations in combination with various C proteins are underway. Supported by NSF 0448676 and the CSUF HHMI Research Scholars Program.