Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 1. Labeling of the enzyme with iodoacetate

A two-step method for labeling the glutamine active site of formyglycinamide ribonucleotide (FGAR) amidotransferase from chicken liver has been developed in which reaction of all other reactive groups with unlabeled iodoacetate is followed by specific labeling of the glutamine site with radioactive...

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Bibliographic Details
Published in:Biochemistry (Easton) 1977-03, Vol.16 (6), p.1065-1069
Main Authors: Ohnoki, Shiro, Hong, Bor-Shyue, Buchanan, John M
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Binding, Competitive
Chickens
Glutamine - analysis
Glycine - analogs & derivatives
Hydrogen-Ion Concentration
Iodoacetates - pharmacology
Kinetics
Ligases - isolation & purification
Ligases - metabolism
Liver - enzymology
Protein Binding
Ribosemonophosphates
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Summary:A two-step method for labeling the glutamine active site of formyglycinamide ribonucleotide (FGAR) amidotransferase from chicken liver has been developed in which reaction of all other reactive groups with unlabeled iodoacetate is followed by specific labeling of the glutamine site with radioactive reagent. A study of the reaction as a function of duration, temperature, and pH of the incubation as well as concentration of iodoacetate has revealed that two nonessential groups of the enzyme react in the presence of glutamine and that this modified enzyme is relatively resistant to further carboxymethylation. When this modified enzyme was incubated with radioactive iodoacetate in the presence of FGAR, ATP, and Mg2+ after removal of glutamine by dialysis, about 1 mol of radioactive iodoacetate was incorporated per mol of enzyme with inactivation. This method permits labeling of the active site for glutamine without the use of glutamine analogues.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00625a006