GLUTAMATE METABOLISM 333 



competitively with respect to phosphate. It was stated that for a substance 

 to compete with glutamine it should have affinity for the enzyme-phosphate 

 complex, and it would seem that glutamate may fall into this category. 



Few other analogs have been tested on this enzyme. Krebs (1935) ob- 

 served a mild inhibition by DL-/?-hydroxyglutamate (22% at 80 mM when 

 glutamine 40 mM), and Girerd et al. (1958) reported inhibition by ethyl 

 D-glutamate and DL-/5-methylglutamate. Because of the postulated role 

 of glutaminase in renal function, these two latter analogs, along with 

 L-glutamate and bromosulfalein, were tested in vivo. These inhibitors re- 

 duce diuresis in rats around 50% at 50 mg/kg subcutaneously, whereas a 

 group of five less potent glutaminase inhibitors actually increase diuresis. 



Formylglycinamidine Phosphoriboside Synthetase 



Glutamine participates in purine biosynthesis by contributing its amide 

 N. Azaserine and 6-diazo-5-oxo-L-norleucine (DON) are potent inhibitors 

 of inosinate biosynthesis and lead to the accumulation of formylglycina- 

 mide phosphoribotide (FGAR). These substances may be considered as 

 analogs of glutamine and have been shown to inhibit formylglycinamide 

 ribonucleotide amidotransferase competitively with respect to glutamine 



(Levenberg et al., 1957). The K„, for glutamine is 0.615 mM and the K^s 

 for azaserine and DON are 0.034 mM and 0.0011 mM, respectively. Once 

 azaserine binds to the enzyme, however, an irreversible reaction occurs, 

 due perhaps to an alkylation of the enzyme. French et al. (1963 a) pointed 

 out that 50% inhibition can be obtained with a (S)/(I) ratio of 2100 with 

 DON. Phosphoribosyl-PP amidotransferase, another enzyme catalyzing 

 the transfer of the amide nitrogen of glutamine, is also inhibited compe- 

 titively by DON, with a K^ of 0.019 mM, and much more weakly by aza- 

 serine (Hartman, 1963 b). A slow covalent binding of DON to the enzyme 

 occurs following the initial reversible attachment, and this is accelerated 

 by the presence of phosphoribosyl-PP and Mg++ on the enzyme, indicating 

 that the active site for the reaction of glutamine, or the binding of DON, 

 is partly dependent on the other substrate and the cofactor. Blocking of 

 an SH group prevents the attachment of DON to the enzyme, suggesting 

 that this SH group is catalytically functional in the nitrogen transfer and 

 the irreversible binding of DON, as French et al. (1963 b) concluded from 

 their work with azaserine on the formylglycinamide ribonucleotide amido- 

 transferase. 



