Table 2.

Stereospecificity of protonation in InhA WT and variants as determined by the label incorporation in the 2S position starting with substrates or with C2-ene adduct

A 200-μl assay contained 400 μm NADH and 300 μm octenoyl-CoA in deuterated 30 mm PIPES, 150 mm NaCl buffer, pD 6.8, and were started with 12.5 μm InhA WT, 22.6 μm InhA Y158F, 70.3 μm Y158S, 23.9 μm InhA T196V, and 40 μm InhA T196A. The reactions were followed spectrophotometrically at 360 nm and run at 30 °C until complete consumption of NADH (∼1 min for WT and 3 h for Y158F, Y158S, and T196A) except for the assay containing T196V, which was stopped after 7 h after approximately 50% of NADH was consumed. Detailed workup of the assay and analysis is described under “Experimental procedures.”

Enzyme variant	No label (2R)	+1 label (2S)
	%	%
InhA WT	99 ± 1	1 ± 1
InhA Y158F	57 ± 2	43 ± 2
InhA Y158S	79 ± 1	21 ± 1
InhA T196A	91 ± 1	9 ± 1
InhA T196V	77 ± 1	23 ± 1