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. Author manuscript; available in PMC: 2010 Apr 14.
Published in final edited form as: Biochemistry. 2009 Apr 14;48(14):3138–3145. doi: 10.1021/bi9000134

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
a

Unable to determine constants due to extremely low activity

b

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

*

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