Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues.
Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.
Zhou
G
G
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Broyles
S S
SS
Dixon
J E
JE
Zalkin
H
H
D000582:Amidophosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D014162:Transfection
D011498:Protein Precursors; D004247:DNA; D000582:Amidophosphoribosyltransferase; D003545:Cysteine