TABLE 1.

Inhibition of wild-type and mutant hGDH1 and hGDH2 by DES in crude extracts

For obtaining the hGDH1 mutants, the GLUD1 cDNA was mutagenized at sites at which hGDH1 differs from hGDH2. In each of these sites, the amino acid residue present in the GLUD2 GDH replaced the corresponding amino acid of the GLUD1 enzyme. For obtaining the S443R-hGDH2 reverse mutant, the GLUD2 cDNA was used as template. Wild-type and mutated cDNAs were expressed in Sf21 cells. Crude homogenates of these cells were used for assaying GDH activity (in the direction of reductive amination of α-ketoglutarate in TRA buffer, pH 8.0) in the absence or in the presence of increasing concentrations of DES. ADP concentration was kept constant at 1 mm. The DES IC₅₀ (± S.E.) values were calculated from representative inhibitory curves for each enzyme using the Origin Program. The number in parentheses represents the number of experimental determinations. p values refer to comparison of the DES IC₅₀ of the wild-type and S443R mutant hGDH2 and of each hGDH1 mutant with that of the wild-type hGDH1 using Student's t test. Mutations in hGDH1 that significantly increased the DES IC₅₀, as well as the wild-type hGDH2 and its S443R mutant, are shown in bold type, whereas the R443S hGDH1 mutation that decreased the DES IC₅₀ is shown in bold and italic type. For each enzyme, two to six additional independent experiments were performed, yielding similar results.

Enzyme	DES IC50 at 1 mm ADP
	μm
WT hGDH1	32.40 ± 1.77 (27)
E34K-hGDH1	24.22 ± 3.24 (24)
R39E-hGDH1	51.52 ± 4.32 (27); <0.01
D142E-hGDH1	29.87 ± 3.15 (24)
I166V-hGDH1	40.92 ± 2.60 (27)
S174N-hGDH1	61.44 ± 3.82 (27); <0.0001
G247R-hGDH1	41.89 ± 3.21 (24)
A321V-hGDH1	44.12 ± 2.90 (24)
S331T-hGDH1	34.85 ± 4.15 (21)
M370L-hGDH1	52.90 ± 3.39 (30); <0.001
M415L-hGDH1	24.29 ± 2.50 (30)
R443S-hGDH1	1.89 ± 0.11 (24); <0.0001
G456A-hGDH1	60.67 ± 3.44 (21); <0.0001
R470H-hGDH1	39.01 ± 2.39 (24)
N498S-hGDH1	26.45 ± 2.35 (26)
WT hGDH2	10.06 ± 1.27 (36); <0.0001
S443R-hGDH2	91.33 ± 9.05 (33); <0.0001