Glutathathione synthetase of bovine lens: anomalies of the enzyme-catalyzed formation of ophthalmic acid.
The activity of glutathione synthetase from bovine lens was examined as a functions of the concentration of L-gamma-glutamyl-L-alpha-aminobutyrate, the dipeptide substrate required in the formation of ophthalmic acid. Several significant anomalies of the glutathione synthetase-catalyzed formation of ophthalmic acid were found. Curvilinearity of double reciprocal plots occurred with this substrate; this curvilinearity shows substrate activation of the reaction which is likely a result of negative cooperativity. Both ATP4- and, to a lesser extent Mg2+ inhibited the reaction, whereas MgATP2- is the substrate; maximum activity occurred with 2 mM Mg2+ in excess of the concentration of added ATP. This investigation shows that it is necessary to establish a defined set of conditions for reporting enzyme activity and that the usual practice of using very large concentrations of Mg2+ relative to ATP, and 5- to 20-fold excess of the dipeptide will give less than optimum activity. The unit of enzyme activity is suggested to be that activity in ml using 2 mM ATP, 4 mM Mg2+, 30 mM glycine and 15 mM L-gamma-glutamyl-alpha-aminobutyrate, which results in the formation of 1 nmole/minute of ADP or P(i). In this study, 5'-AMP was for the first time, shown to be an inhibitor of the reaction with a K(i) of 0.9 mM.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004151:Dipeptides / Q000378:metabolism*; D005981:Glutathione Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism*; D013379:Substrate Specificity
D004151:Dipeptides; C042262:gamma-glutamyl-alpha-aminobutyrate; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; D005981:Glutathione Synthase; D008274:Magnesium