Table 5.

Kinetic characterization of erythrose reduction activity of human recombinant ADH1 variant proteins and SORD in vitro using NADPH as the cofactor

Reaction buffer consisted of 50 mm sodium phosphate, pH 7.2, 200 μm NADPH or NADH, and at least 5 concentrations of erythrose ranging from 17.5 to 1120 mm. Reactions were initiated with addition of 100 nm purified protein. The rate of NADPH or NADH consumption at 340 nm was monitored using a Shimadzu UV-2600 spectrophotometer. Measurements at each substrate concentration were performed in duplicate (two technical replicates). For ADH1B2, ADH1C2, and SORD, the K_m and V_max values from each independent experiment was determined by fitting the data to Y = V_max × X/(K_m + X), where Y represents reaction velocity and X indicates substrate concentration using GraphPad Prism software. For ADH1B1, the K_m,V_max, and K_i values from each independent experiment was determined by fitting the data to Y = V_max × X/(K_m + X × (1 + X/K_i)), where Y represents reaction velocity and X indicates substrate concentration using GraphPad Prism software. Data are shown as average and S.D. of two independent experiments. K_m values are presented as “apparent K_m” because these values were determined at only one saturating concentration of cofactor.

Allele	Highest prevalence	Apparent Km	kcat	kcat/Km	Substrate inhibition
		mm	s−1	m−1 s−1
NADPH used as cofactor
ADH1B1	Caucasian, African American	322 ± 168	0.9 ± 0.3	1.4 ± 1.2	Ki = 335 ± 237 mm
ADH1B2	Asian	No activity detected	No activity detected	No activity detected	No activity detected
ADH1C2	Caucasian	518 ± 180	0.40 ± 0.07	0.9 ± 0.3	None
SORD		118 ± 11	1.58 ± 0.05	13.8 ± 1.4	None
NADH used as cofactor
ADH1B1	Caucasian, African American	272 ± 70	1.04 ± 0.19	3.8 ± 1.2	Ki = 403 ± 127 mm
ADH1B2	Asian	228 ± 36	0.70 ± 0.05	3.1 ± 0.6	None
ADH1C2	Caucasian	443 ± 78	1.60 ± 0.17	3.6 ± 0.7	None
SORD		94 ± 2	4.10 ± 0.03	43.1 ± 2.0	None