Identification of three key active site residues in the C-terminal domain of human recombinant folylpoly-gamma-glutamate synthetase by site-directed mutagenesis.
Three cysteines in human recombinant folylpoly-gamma-glutamate synthetase (FPGS) that were reactive with iodoacetamide were located in peptides that were highly conserved across species; the functions of two of these peptides, located in the C-terminal domain, were studied by site-directed mutagenesis. When cDNAs containing mutations in each conserved ionic residue on these peptides were transfected into AUXB1 cells, which lack endogenous FPGS activity, one mutant (D335A) did not complement the auxotrophy, and another (R377A) allowed only minimal growth. FPGS activity could not be detected in insect cells expressing abundant levels of these two mutant proteins from recombinant baculoviruses nor from a virus encoding an H338A mutant FPGS. Kinetic analysis of the purified proteins demonstrated that each of these three mutants was quite different from the others. The major kinetic change detected for the H338A mutation was a 600-fold increase in the K(m) for glutamic acid. For the D335A mutation, the binding of all three substrates (aminopterin, ATP, and glutamic acid) was affected. For R377A, the K(m) for glutamic acid was increased by 1500-fold, and there was an approximately 20-fold decrease in the k(cat) of the reaction. The binding of the K(+) ion, a known activator of FPGS, was affected by the D335A and H338A mutations. We conclude that these three amino acids participate in the alignment of glutamic acid in the active site and that Arg-377 is also involved in the mechanism of the reaction.
Sanghani
S P
SP
Department of Pharmacology and Toxicology and the Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Sanghani
P C
PC
Moran
R G
RG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D011188:Potassium / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D011188:Potassium; D007460:Iodoacetamide