Liu Y, Vanhooke J L, Frey P A
Institute for Enzyme Research, Graduate School, Madison, Wisconsin 53705, USA.
Biochemistry. 1996 Jun 11;35(23):7615-20. doi: 10.1021/bi960102v.
UDP-galactose 4-epimerase from Escherichia coli contains tightly bound NAD+, which participates in catalyzing the interconversion of UDP-galactose and UDP-glucose through its redox properties. The purified enzyme is a dimer of identical subunits that consists of a mixture of catalytically active subunits designated E.NAD+ and inactive, abortive complexes designated E.NADH.uridine nucleotide, in which the uridine nucleotide may be UDP-glucose, UDP-galactose, or UDP [Vanhooke, J. L., & Frey, P. A. (1994) J. Biol. Chem. 269, 31496-31404]. The abortive complexes are transformed into active E.NAD+ by denaturation of the purified enzyme at 4 degrees C in 6 M guanidine hydrochloride buffered at pH 7.0 in the presence of 0.126 mM NAD+ for 3 h, followed by dilution of guanidine hydrochloride to 0.18 M and of NAD+ to 0.076 mM for 2 h. The renatured enzyme is fully active and contains negligible amounts of NADH and uridine nucleotides. The extinction coefficent of the epimerase at 280 nm is 1.81 +/- 0.15 mL mg-1 cm-1 (epsilon 280 = 137 +/- 11 mM-1 cm-1), as determined by quantitative amino acid analysis and spectrophotometric measurements. This value allows the value of the extinction coefficient for the reduced enzyme (E.NADH)to be calculated as epsilon 344 = 5.7 mM-1 cm-1. On the basis of the new value of epsilon 280, analytical measurements of the nAD+ content of epimerase show that there are two molecules of NAD+ per dimer, which confirms conclusions from X-ray crystallography and revises the earlier bioanalytical determinations. The ultraviolet/visible absorption spectrum of E.NAD+ from denaturation-renaturation experiments reveals the presence of a broad absorption band extending from 300 nm to beyond 360 nm that cannot be attributed to NADH and appears to be a charge-transfer band. This band is partially bleached by UMP and almost totally abolished by UDP, indicating that the interactions leading to the charge-transfer band are altered by the uridine nucleotide-induced conformational change in this enzyme. This conformational change is associated with control of the chemical reactivity of NAD+ in the reaction mechanism.
来自大肠杆菌的UDP-半乳糖4-表异构酶含有紧密结合的NAD⁺，它通过其氧化还原特性参与催化UDP-半乳糖和UDP-葡萄糖的相互转化。纯化后的酶是由相同亚基组成的二聚体，由具有催化活性的亚基（称为E.NAD⁺）和无活性的、流产型复合物（称为E.NADH·尿苷核苷酸）混合而成，其中尿苷核苷酸可能是UDP-葡萄糖、UDP-半乳糖或UDP [范胡克，J. L.，& 弗雷，P. A.（1994年）《生物化学杂志》269卷，31496 - 31404页]。在0.126 mM NAD⁺存在下，于pH 7.0缓冲的6 M盐酸胍中，将纯化后的酶在4℃变性3小时，然后将盐酸胍稀释至0.18 M，NAD⁺稀释至0.076 mM并持续2小时，流产型复合物就会转化为有活性的E.NAD⁺。复性后的酶具有完全活性，且NADH和尿苷核苷酸的含量可忽略不计。通过定量氨基酸分析和分光光度测量确定，表异构酶在280 nm处的消光系数为1.81 ± 0.15 mL mg⁻¹ cm⁻¹（ε280 = 137 ± 11 mM⁻¹ cm⁻¹）。该值使得还原型酶（E.NADH）的消光系数值可计算为ε344 = 5.7 mM⁻¹ cm⁻¹。基于ε280的新值，对表异构酶中NAD⁺含量的分析测量表明，每个二聚体中有两个NAD⁺分子，这证实了X射线晶体学的结论，并修正了早期的生物分析测定结果。变性 - 复性实验中E.NAD⁺的紫外/可见吸收光谱显示，存在一个从300 nm延伸至360 nm以上的宽吸收带，该吸收带不能归因于NADH，似乎是一个电荷转移带。该吸收带被UMP部分漂白，被UDP几乎完全消除，这表明导致电荷转移带的相互作用因该酶中尿苷核苷酸诱导的构象变化而改变。这种构象变化与反应机制中NAD⁺化学反应性的控制有关。
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