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. Author manuscript; available in PMC: 2010 Dec 6.
Published in final edited form as: Structure. 2005 Jun;13(6):929–942. doi: 10.1016/j.str.2005.03.018

Table 2.

Activity of ArnA Dehydrogenase Mutants

Enzyme Rate,
nmolNADH·mgEz−1·min−1 		Fold
Decrease
in Activity
Wild-type ArnA _DH 215.9 ± 0.4 –
S433A (1 mM UDP-GlcA) 6.33 ± 0.03 30
S433A (5 mM UDP-GlcA) 6.43 ± 0.10 30
R619M (1 mM UDP-GlcA) 0.26 ± 0.05 800
R619M (5 mM UDP-GlcA) 0.51 ± 0.10 400
R619Y (1 mM UDP-GlcA) 0.02 ± 0.02 >10000
S433T (1 mM UDP-GlcA) 0.01 ± 0.02 >10000
R619E (1 mM UDP-GlcA) 0.00 ± 0.02 >10000

The activity of the mutants was measured spectrophotometrically by detecting the increase of absorbance at 340 nm due to the release of NADH. The substrates UDP-GlcA and NAD+ were used at saturating concentrations (UDP-GlcA, 1 or 5 mM; NAD, 3 mM) for the wild-type enzyme (Km, app [NAD] = 0.57 ± 0.09 mM; Km, app [UDP-GlcA] = 0.054 ± 0.003 mM [Gatzeva-Topalova et al., 2004]).