Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP

Abstract

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-a-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992-->Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954-->Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch-Nyhan syndrome

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

“…The mutation T974 to A974 disrupted the IMP/UMP binding pocket in E. coli CPS and not only abolished UMP inhibition and IMP activation, but also decreased activation by ornithine (Fresquet et al, 2000). Interestingly, this site also plays a role in allosteric control in mammalian CPSII as well since mutation of the corresponding hamster residue S1355 to A1355 nearly abolished activation by PRPP and lowered overall CPSII activity ~5-fold (Simmons et al, 2004). While T. gondii CPSII is nonresponsive to PRPP in vitro , mutation of the analogous residue in T. gondii CPSII (T1530 to A1530) significantly reduced CPSII complementation activity based on a reduced initial parasite growth rate (from 7.4 to 8.6 h), and reduced transient complementation efficiency to 51% and stable complementation efficiency to 10% of the control suggesting several copies of this mutant allele are necessary to fully support parasite growth (Table 3).…”

“…While the structure, function, and mechanisms of CPS catalysis and regulation have been well characterized for E. coli CPS (Thoden et al, 1997; Mora et al, 1999; Thoden et al, 1999b; Fresquet et al, 2000; Huang and Raushel, 2000a, 2000b, 2000c; Miles and Raushel, 2000), the larger CPSII polypeptides of eukaryotes are not yet well characterized at both the functional and structural level (Davidson et al, 1993; Graves et al, 2000; Fox and Bzik, 2003; Simmons et al, 2004; Kothe et al, 2005). Here, we report functional complementation of uracil auxotrophy in T. gondii based on CPSII minigenes.…”