Studies on Ligand Binding and Catalysis by Isoleucine Epimerase and CTP Synthase

Abstract

Isoleucine 2-epimerase participates in production of branched-chain D-amino acids. We showed that L- and D-neopentyl glycine are substrates of LbIleE, with Km values of 0.65 ± 0.03 mM and 0.43 ± 0.03 mM, respectively. The corresponding kcat/Km values (1.45 ± 0.04 mM–1s–1 and 1.58 ± 0.14 mM–1s–1) were reduced ~ 45-fold relative to that of L-Ile. The fluorinated derivative NV-5138 was also shown to be a substrate, with the fluorines having little effect on binding. CTP synthase catalyzes the formation of CTP from UTP. We constructed EcCTPS lid L11 variants: G357A, G357V, F353Y, F353H, F353W, and F353L. All variants were fully active with exogenous NH3 but exhibited decreased catalytic and activation efficiency with Gln and GTP (WT ~ G357A > G357V, WT > F353Y > F353H > F353W and F353L). NH3 transport and GTP binding were unaffected; however, the glutaminase activity was markedly reduced.