Search

Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.

In vitro experiments showed that 6-diazo-5-oxo-L-norleucylglycine, a dipeptide analog of L-glutaminylglycine, inactivates gamma-glutamyl transferase bound to renal brush border membrane vesicles but does not inactivate the purified transferase. The rate of inactivation of the membrane-bound enzyme decreased markedly in the presence of dipeptides, such as L-leucylglycine and L-alanylglycine, or in the presence of o-phenanthroline, an inhibitor of renal peptidases. The presence of L-cysteinylglycine S-acetyldextran polymer (Mr 500,000), which does not permeate membranes, protected the membrane-bound transferase from inactivation by 6-diazo-5-oxo-L-norleucyglycine. This and other findings suggest that the norleucylglycine derivative was hydrolyzed by peptidase(s) bound to the outer surface of the brush border membranes and that the 6-diazo-5-oxo-L-norleucine thus released acts as an affinity-labeling reagent for the membrane-bound transferase. Similar effects were observed in vivo. Intravenous administration of 6-diazo-5-oxo-L-norleucylglycine to mice resulted in a marked decrease in renal transferase activity. Mice thus pretreated with 6-diazo-5-oxo-L-norleucylglycine, but not an untreated group, excreted significant amounts of S-carbamido[14C]methylglutathione in their urine within 30 min of intravenous administration of this compound. This finding suggests that the renal transferase was involved in the hydrolysis of the glutathione S-conjugate in the glomerular filtrate in vivo and that the administered 6-diazo-5-oxo-L-norleucylglycine underwent hydrolysis peptidase(s)-catalyzed to liberate 6-diazo-5-oxo-L-norleucine that reacted with the membrane-bound gamma-glutamyl transferase.