Table 1. Steady-State Kinetic Parameters for the Decarboxylase and Oxidase Activities of C-Terminally His₆-Tagged, WT OxDC and Selected OxDC Loop Variants.

		decarboxylase activityb			oxidase activityc
enzyme	Mn/monomera	KM (mM)	kcat (s–1)	kcat/KM/Mn (M–1 s–1)	KM (mM)	kcat (s–1)	kcat/KM/Mn (M–1 s–1)	specificity switchd
WT OxDC	1.4	8 ± 1	60 ± 2	5700 ± 840	5.0 ± 0.2	0.13 ± 0.02	19 ± 3	1
DASNe	1.6	16 ± 7	0.4 ± 0.1	17 ± 8	3.0 ± 0.3	4.1 ± 0.4	800 ± 190	14100 (225000)f
DESNe	1.5	6 ± 1	40 ± 1	4400 ± 660	11 ± 2	0.10 ± 0.01	11 ± 1	0.75 (9)
DENS	1.6	10 ± 1	63 ± 3	3900 ± 840	4.0 ± 0.4	0.05 ± 0.01	9 ± 2	1
DDSN	1.5	3 ± 1	9 ± 2	2400 ± 910	17 ± 2	0.79 ± 0.03	32 ± 4	2
DDNS	1.5	10 ± 2	45 ± 3	2900 ± 680	16 ± 2	0.49 ± 0.04	20 ± 3	32
SDNSg	1.1	3.1 ± 0.7	29 ± 2	9000 ± 200	ndi	ndi	ndi	ndi
SQNSg	0.5	10.0 ± 0.2	0.3 ± 0.1	60 ± 20	ndi	ndi	ndi	ndi
SANSh	1.4	ndi	inactive	ndi	ndi	ndi	ndi	ndi
ΔE162	1.3	1.6 ± 0.7	0.01 ± 0.005	5 ± 2	3 ± 1	0.05 ± 0.03	14 ± 9	840

^aThe metal content value has been reported previously for WT OxDC and is included here for ease of comparison.¹⁷

^bReaction mixtures consisted of WT OxDC or the loop variant (5 μM) and potassium oxalate (0–80 mM) dissolved in 100 mM citrate buffer containing 300 μM o-phenylenediamine (pH 4.2) at 25 °C (total volume of 1 mL). Turnover was initiated by the addition of enzyme. After incubation at 25 °C for 1 min, the reaction was quenched by the addition of 1.1 M aqueous NaOH (10 μL) and the formate quantified using the NADH absorption at 340 nm in an assay mixture containing formate dehydrogenase (pH 7.8).¹⁷

^cReaction mixtures consisted of WT OxDC or the loop variant (0.5 μM) and potassium oxalate (0–50 mM) dissolved in 50 mM succinate buffer (pH 4.0) at 25 °C (total volume of 1 mL). Turnover was initiated by the addition of enzyme, and hydrogen peroxide was quantified using the horseradish peroxidase–ABTS assay. Initial rates were determined from the linear portion of the continuous assay.²⁰

^dThe specificity switch value is calculated using eq 1 (see Materials and Methods).

^eSteady-state kinetic parameters have been reported previously for this OxDC loop variant.¹⁴ The values reported herein, however, have been redetermined for the recombinant enzyme expressed in our laboratory using reaction conditions identical to those reported in the earlier study.

^fThe specificity switch value in parentheses is that reported previously and is included in the table for ease of comparison. We note, however, that this earlier values was calculated using the following expression,¹⁴ which differs from the equation (eq 1) used in our study: {[^MUT(V_max)_OX][^WT(V_max)_DEC]}/{[^MUT(V_max)_DEC][^WT(V_max)_OX]} (eq 2), where ^MUT(V_max)_OX and ^MUT(V_max)_DEC are the V_max values for the oxidase and decarboxylase activities of the OxDC loop variant, respectively. Similarly, ^WT(V_max)_OX and ^WT(V_max)_DEC are the cognate values of WT OxDC.

^gValues taken from ref (21) are included for ease of comparison. The specific activities reported for the oxidase activity of the SDNS and SQNS OxDC variants were 0.05 and 0.56 unit/mg, respectively. Note that both of these variants lacked a C-terminal His tag, and the assay conditions were different from those employed in this study.

^hValues taken from ref (27) are included for ease of comparison. The specific activity reported for the oxidase activity of the SANS OxDC variant was 0.02 unit/mg. This variant was reported to lack detectable decarboxylase activity.

ⁱNot determined.