TABLE 2.

Steady-state kinetic constants for Salmonella serovar Typhimurium AEPT mutants

AEPTa	Km of AEP (mM)	Km of pyruvate (mM)	Km of P-Ald (mM)	Km of l-Ala (mM)	kcat (s−1)→P-Aldb	kcat (s−1)→AEPc
Wild type	1.11 ± 0.03	0.150 ± 0.02	0.09 ± 0.01	1.4 ± 0.3	7.4	9.3
D168A	NAd	NA	NA	NA	NA	≤1 × 10−4
K194R	NA	NA	NA	NA	NA	≤1 × 10−4
K194L	NA	NA	NA	NA	NA	≤1 × 10−4
R340K	−e	0.50 ± 0.05	2.9 ± 0.4	20 ± 1	0.2	0.6
R340A	26 ± 1	6.1 ± 0.3	0.19 ± 0.01	140 ± 70	0.2	0.2

^aThe 1-ml assay solution for the direction of P-Ald formation contains 0.5 mM β-NADH, 100 μM PLP, 10 U of ADH, 2 U of phosphonatase, and 5 mM MgCl₂ in 50 mM K⁺TRICINE (pH 8.5) at various concentrations of AEP (5 to 40 mM) with 5 mM pyruvate or various concentrations of pyruvate (0.4 to 7 mM) with 20 mM AEP. The 1-ml assay solution for the direction of AEP formation contains 0.5 mM β-NADH, 100 μM PLP, 10 U of LDH, and 5 mM MgCl₂ in 50 mM K⁺TRICINE (pH 8.5) at various concentrations of P-Ald (0.25 to 10 mM) with 20 mM l-Ala or various concentrations of l-Ala (3 to 20 mM) with 5 mM P-Ald.

^b→P-Ald, in the direction of P-Ald formation.

^c→AEP, in the direction of AEP formation.

^dNA, no activity.

^eThe K_m was too large to measure.