Temperature-dependent function of the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel and coupling with glycinamide ribonucleotide synthetase in a hyperthermophile

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT ) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus we re expressed in Escherichia coli, and the enzymes were purified to near hom ogeneity, Both enzymes were maximally active at a temperature of at least 9 0 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 deg rees C, GPAT activity is known to depend upon channeling of NH, from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphat e site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH, for synthesis of PRA was f ound to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C. The mechanism for transfer of PRA to GARS is not established, but diffu sion between enzymes as a free intermediate appears unlikely based on a cal culated PRA half-life of approximately 0.6 s at 90 degrees C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50 degree s C, and remained relatively constant up to 90 degrees C. The calculated PR A chemical half-life, however, decreased by a factor of 20 over this temper ature range. These results provide evidence that coupling involves direct P RA transfer through GPAT-GARS interaction rather than free diffusion.