Two 4-fluoro-L-glutamine diastereoisomers [(2S,4R)-4-FGln, (2S,4S)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with, [F-18(2S,4R)-4-FGln than with [F-18](2S,4S)-4-FGln. In the present work we investigated the enzymology of two diastereoisomers of 4-FGln, two diastereoisomers of 4-fluoroglutamate (4-FGlu) (potential metabolites of the 4-FGln diastereoisorners) and another fluoro-derivative of L-glutamine [(2S,4S)-4-(3-fluoropropyl) glutamine (FP-Gln)]. The two 4-FGlu diastereoisomers were found to be moderate-to-good substrates relative to L-glutamate of glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. Additionally, alanine aminotransferase was shown to catalyze an unusual gamma-elimination reaction with both 4-FGlu diastereoisomers. Both 4-FGlu diastereoisomers were shown to be poor substrates, but strong inhibitors of glutamine synthetase. Both 4-FGln diastereoisomers were shown to be poor substrates compared to L-glutamine of glutamine transaminase L and alpha-arninoadipate aminotransferase. However, (2S,4R)-4-FGln was found to be a poor substrate of glutamine transaminase K, whereas (25,4S)-4-FGln was shown to be an excellent substrate. By contrast, FP-Gln was found to be a poor substrate of all enzymes examined. Evidently, substitution of H in position 4 by F in L-glutamine/L-glutamate has moderate-to-profound effects on enzyme-catalyzed reactions. The present results: 1) show that 4-FGIn and 4-FGlu diastereoisomers may be useful for studying active site topology of glutamate- and glutamine-utilizing enzymes; 2) provide a framework for understanding possible metabolic transformations in tumors of F-18-labeled (2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu or (2S,4S)-4-FGlu; and 3) show that [F-18]FP-Gln is likely to be much less metabolically active in vivo than are the [F-18]-FGln diastereoisomers. (C) 2016 Elsevier B.V. All rights reserved.