Table 1. Steady-state kinetic properties of E. coli AHAS isozyme II and some derivatives.

	Specific act,†	Km for pyruvate,†		kcat/Km,†			K0.5 for ThDP,†
Construct*	units/mg	mM	kcat,† s-1	M-1·s-1	R‡	Vb/Va§	μM
WT	20	5.2	23.3	4,490	56 ± 7	1.0	0.63
His′-WT	17	6	19.8	3,305	59 ± 3	1.0	1.2
His′-Met250Ala	2.6	36	3.03	84	56	8.0	1.2
His′-Trp464Leu	11.9	11.2	13.9	1,240	3.0 ± 0.2	1.0	1.7
His′-Arg276Lys	2.8	38	3.27	85	28	4.0	2

^*The WT AHAS and the mutated enzymes were expressed and purified either as the natural holoenzyme (4) or as a protein with an N-terminal hexahistidine fusion (His′-) (11). Some of this data has appeared in a previous publication (11)

^†These kinetic parameters were determined in steady-state experiments using the colorimetric assay for acetoin (17), with 100 mM pyruvate as the sole substrate, in the presence of 0.1 M KPi buffer (pH 7.6) containing 10 mM MgCl₂, 0.1 mM ThDP, and 75 μM FAD, except for variation of the relevant reactant. k_cat is defined as the turnover rate per site, assuming two sites in the 138-kDa heterotetramer. The parameters have experimental uncertainties of 5–10%

^‡R is the substrate specificity for 2-ketobutyrate as second substrate, (AHB formed)/(AL formed) = R × [2-ketobutyrate]/[pyruvate], and was determined by measuring AHB and AL formation simultaneously (8) in a competition experiment with varying 2-ketobutyrate and 50 mM pyruvate, in 0.1 M KPi buffer (pH 7.6) containing 10 mM MgCl₂, 0.1 mM ThDP, and 75 μM FAD (11)

^§V_b/V_a is the ratio between the rate of AL formation with 50 mM pyruvate as sole substrate and the extrapolated rate of AHB formation in the presence of 50 mM pyruvate at saturation with 2-ketobutyrate. The experimental uncertainty in V_b/V_a is 10–15%