Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP.
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
Simmons
Christine Q
CQ
Department of Microbiology, Immunology and Molecular Genetics, Albert B. Chandler Medical Center and Lucille P. Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0293, USA.
Simmons
Alan J
AJ
Haubner
Aaron
A
Ream
Amber
A
Davidson
Jeffrey N
JN
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004080:Dihydroorotase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010754:Phosphoribosyl Pyrophosphate / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D014544:Uridine Triphosphate / Q000494:pharmacology*
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D012694:Serine; D010754:Phosphoribosyl Pyrophosphate; D014364:Tryptophan; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008239:Lysine; D014544:Uridine Triphosphate