Glycinamide ribonucleotide synthetase from Escherichia coli: cloning, overproduction, sequencing, isolation, and characterization.
The purD gene of Escherichia coli encoding the enzyme glycinamide ribonucleotide (GAR) synthetase, which catalyzes the conversion of phosphoribosylamine (PRA), glycine, and MgATP to glycinamide ribonucleotide, MgADP, and Pi, has been cloned and sequenced. The protein, as deduced by the structural gene sequence, contains 430 amino acids and has a calculated Mr of 45,945. Construction of an overproducing strain behind a lambda pL promoter allowed a 4-fold purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of the sequence with those of other GAR synthetases confirm the amino acid sequence deduced from the gene sequence. Initial velocity studies and product and dead-end inhibition studies are most consistent with a sequential ordered mechanism of substrate binding and product release in which PRA binds first followed by MgATP and then glycine; Pi leaves first, followed by loss of MgADP and finally GAR. Incubation of [18O]glycine, ATP, and PRA results in quantitative transfer of the 18O to Pi. GAR synthetase is very specific for its substrate glycine.
Cheng
Y S
YS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Shen
Y
Y
Rudolph
J
J
Stern
M
M
Stubbe
J
J
Flannigan
K A
KA
Smith
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005786:Gene Expression Regulation; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D010103:Oxygen Isotopes; D010758:Phosphorus / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D012267:Ribosemonophosphates; C008891:phosphoribosylamine; D010758:Phosphorus; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022387:phosphoribosylamine-glycine ligase; D005998:Glycine