Table 2.

Kinetic parameters of LrPduO wild type and variants using the Co²⁺ assay. The substrate cob(II)alamin was generated using a protein reducing system

Enzyme	ATP			Cob(II)alamin
	Km (μM)	kcat (s−1)	kcat/Km (M−1 s−1)	Km (μM)	kcat (s−1)	kcat/Km (M−1 s−1)
wild type	5.5 ± 1.1	(2.9 ± 0.1) × 10−2	(5.5 ± 1.1) × 103	7.8 ± 1.1	(3.8 ± 0.5) × 10−2	(4.8 ± 0.9) × 103
F112A	UDa	UD		UD	UD
F112H	10.4 ± 0.8	(5.9 ± 0.2) × 10−4	(5.7 ± 0.5) × 101	53.0 ± 8.6	(6.7 ± 0.2) × 10−4	(1.3 ± 0.2) × 101
F112Y	9.4 ± 0.4	(2.3 ± 0.1) × 10−3	(2.5 ± 0.2) × 102	34.2 ± 10.7	(2.7 ± 0.4) × 10−3	(8.3 ± 2.8) × 101
F112W	7.3 ± 0.1	(2.8 ± 0.1) × 10−3	(3.9 ± 0.2) × 102	28.8 ± 5.7	(3.2 ± 0.1) × 10−3	(1.1 ± 0.2) × 102
F163A	96.1 ± 7.1b	(1.5 ± 0.1) × 10−2 b	(1.6 ± 0.2) × 102 b	134 ± 13	(2.7 ± 0.4) ×10−2	(2.0 ± 0.3) × 102
F187A	7.9 ± 1.0	(1.4 ± 0.1) × 10−2	(1.8 ± 0.3) × 103	15.8 ± 3	(1.5 ± 0.1) × 10−2	(9.3 ± 1.9) × 102
ΔS183	9.9 ± 0.9	(1.7 ± 0.1) × 10−2	(1.7 ± 0.2) × 103	16.3 ± 1.7	(1.8 ± 0.1) × 10−2	(1.1 ± 0.1) × 103

^aUnable to determine constants due to extremely low activity

^bKinetic parameters obtained under sub-saturating concentrations of cob(II)alamin

^*Saturating ATP was at least 100-fold the K_M, however saturating cob(II)alamin was limited to 3- to 10- fold the K_M as a result of assay sensitivity.