Sharma N, Phutela A, Malhotra S P., Singh R
Department of Biochemistry, Plant Biochemistry and Molecular Biology Laboratory, CCS, Haryana Agricultural University, 125 004, Hisar, India
Plant Sci. 2001 Mar;160(4):603-610. doi: 10.1016/s0168-9452(00)00421-0.
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.
磷酸二羟丙酮还原酶（DHAP还原酶）通过硫酸铵分级沉淀、亲和色谱、凝胶过滤和吸附色谱从油菜未成熟种子中纯化至表观电泳纯，回收率约为24%。纯化后的酶分子量约为62 kDa，是一种亚基分子量为32 kDa的二聚体。该酶在pH 7.5时表现出最大活性，对NADH和DHAP具有高度特异性。两种底物均呈现典型的米氏动力学，NADH和DHAP的K(m)值分别为3.3和26.6 μM。该酶的活性不需要任何金属离子。相反，其活性受到Na(+)、K(+)、Mn(2+)、Mg(2+)和Ca(2+)的抑制。ATP和果糖-1,6-P(2)对DHAP的抑制作用为非竞争性，K(i)值分别为0.96和1.3 mM。底物相互作用动力学和产物抑制研究与强制有序的双底物双产物反应机制一致，其中NADH是第一个结合的底物，NAD是最后一个解离的产物。基于此处讨论的特性，该酶可能在由DHAP生成甘油-3-P的过程中发挥作用。
