Rawat U B, Rao M B
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Biochim Biophys Acta. 1996 Apr 16;1293(2):222-30. doi: 10.1016/0167-4838(95)00249-9.
Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.
粗糙脉孢菌的木糖还原酶（XR）被纯化至同质，发现其对NADPH（烟酰胺腺嘌呤二核苷酸磷酸）具有特异性。通过凝胶过滤和SDS-PAGE分析，纯化后的酶显示分子量分别为60 kDa和29 kDa，表明存在两个亚基。木糖还原酶的动力学机制为“等序双双反应”。发现N-溴代琥珀酰亚胺（NBS）对XR的失活呈双相性，快速相（kf）和慢速相（ks）的二级反应速率常数分别为2.5×10²和80 M⁻¹s⁻¹。NADPH可保护90%的XR活性免受NBS的抑制。荧光和圆二色性（CD）研究表明，失活并非由于酶的总体构象变化。对修正的Stern-Volmer图的分析表明，49%的色氨酸荧光可被淬灭，在NADPH存在下这种淬灭完全消除，证实色氨酸参与辅酶结合位点。此处提供的实验证据表明低亲和力NADPH结合位点存在色氨酸残基，并且已通过使用疏水探针ANS评估了该位点。
