Nakanishi M, Kakumoto M, Matsuura K, Deyashiki Y, Tanaka N, Nonaka T, Mitsui Y, Hara A
Department of Applied Chemistry, Faculty of Engineering, Gifu University.
J Biochem. 1996 Aug;120(2):257-63. doi: 10.1093/oxfordjournals.jbchem.a021407.
Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, shows a strong coenzyme preference for NADP(H) over NAD(H), and is uniquely activated by fatty acids. Previous chemical modification and X-ray crystallography studies show that interactions responsible for the coenzyme specificity include salt linkages between the 2'-phosphate of NADPH and side-chains of Lys-17 and Arg-39 of the enzyme. Although Arg-39 is highly conserved in NADP(H)-dependent enzymes of the SDR family, Lys-17 is substituted with Arg in about half of the NADP(H)-dependent enzymes. The present study shows that mutations of Lys-17 to His (K17H) or Ser (K17S) and of Arg-39 to Ala (R39A) bring about decreases (from 5 to 90-fold) of the affinities for NADP(H), but minor changes in the affinity for NAD+. The binding energy arising from the mutations on the binding of the 2'-phosphate of NADP+ was decreased by 38-66% from the value of 4.8 kcal/mol calculated for the wild-type enzyme. In contrast, the mutation of Lys-17 to Arg (K17R) had little effect on the kinetic or thermodynamic properties. The activation by fatty acids was completely attenuated by the mutations of K17H and K17S, but not by K17R or R39A. These results indicate that the 2'-phosphate group of NADP(H) is recognized by both Lys-17 and Arg-39, of which Lys-17 is a component of the binding site for the activator, probably interacting with the negatively charged carboxylate group of fatty acids, and also suggest that the existence of a positively charged residue (either Lys or Arg) at position 17 is required for both NADP(H) specificity of the SDR family enzymes and fatty acid activation of the pulmonary carbonyl reductase.
小鼠肺羰基还原酶是短链脱氢酶/还原酶（SDR）家族的成员之一，对烟酰胺腺嘌呤二核苷酸磷酸（NADP(H)）的辅酶偏好性远高于烟酰胺腺嘌呤二核苷酸（NAD(H)），并且独特地被脂肪酸激活。先前的化学修饰和X射线晶体学研究表明，负责辅酶特异性的相互作用包括NADPH的2'-磷酸基团与该酶的赖氨酸-17（Lys-17）和精氨酸-39（Arg-39）侧链之间的盐键。尽管Arg-39在SDR家族的NADP(H)依赖性酶中高度保守，但在大约一半的NADP(H)依赖性酶中，Lys-17被Arg取代。本研究表明，Lys-17突变为组氨酸（K17H）或丝氨酸（K17S）以及Arg-39突变为丙氨酸（R39A）会导致对NADP(H)的亲和力降低（5至90倍），但对NAD+的亲和力变化较小。NADP+的2'-磷酸基团结合时因突变产生的结合能比野生型酶计算得到的4.8千卡/摩尔的值降低了38 - 66%。相比之下，Lys-17突变为Arg（K17R）对动力学或热力学性质影响很小。K17H和K17S的突变完全消除了脂肪酸的激活作用，但K17R或R39A的突变则没有。这些结果表明，NADP(H)的2'-磷酸基团同时被Lys-17和Arg-39识别，其中Lys-17是激活剂结合位点的组成部分，可能与脂肪酸带负电荷的羧基相互作用，这也表明SDR家族酶的NADP(H)特异性和肺羰基还原酶的脂肪酸激活都需要在第17位存在带正电荷的残基（Lys或Arg）。
