Purification and characterization of dihydroxyacetone phosphate reductase from immature seeds of Brassica campestris L.

Dihydroxyacetone phosphate reductase (DHAP reductase) was purified to apparent electrophoretic homogeneity with about 24% recovery from immature seeds of Brassica campestris using (NH(4))(2)SO(4) fractionation, affinity chromatography, gel filtration and adsorption chromatography. The purified enzyme with molecular mass of about 62 kDa was a dimer with subunit molecular mass of 32 kDa. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for NADH and DHAP. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 3.3 and 26.6 &mgr;M for NADH and DHAP, respectively. The enzyme did not require any metal ion for its activity. Rather, the activity was inhibited by Na(+), K(+), Mn(2+), Mg(2+,) and Ca(2+). ATP and fructose-1,6-P(2) inhibited the enzyme non-competitively with respect to DHAP with K(i) values of 0.96 and 1.3 mM, respectively. Substrate interaction kinetics and product inhibition studies were consistent with compulsory-ordered bi-bi reaction mechanism with NADH being the first substrate to bind and NAD being the last product to dissociate. Based on the properties discussed here, it appears that the enzyme probably functions for the production of glycerol-3-P from DHAP.