Huang Yu Chu, Colman Roberta F
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2002 Apr 30;41(17):5637-43. doi: 10.1021/bi0200716.
Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.
序列比对预测，猪心NADP依赖性异柠檬酸脱氢酶的His(309)等同于大肠杆菌该酶的His(339)，后者在晶体结构中与辅酶相互作用[赫尔利等人（1991年），《生物化学》30, 8671 - 8688]，并且猪His(315)和His(319)靠近该位点。通过定点诱变制备了突变型猪酶H309Q、H309F、H315Q和H319Q，在大肠杆菌中表达并纯化。H319Q突变体对NADP、异柠檬酸和Mn(2+)的K(m)值与野生型酶相似，V(max)=20.1，而野生型为37.8微摩尔NADPH·分钟(-1)·(毫克蛋白质)(-1)。因此，His(319)不参与辅酶结合，对催化作用影响最小。相比之下，H315Q对NADP的K(m)值是野生型的40倍，V(max)=16.2单位/毫克蛋白质，对异柠檬酸和Mn(2+)的K(m)值与野生型相似。这些结果表明His(315)在NADP位点区域起作用。用Q或F取代His(309)得到的酶无检测到的活性。His(309)突变体在野生型和H319Q结合1.0摩尔NADPH/摩尔亚基的条件下，对NADPH的结合很差，表明His(309)对辅酶结合很重要。His(309)突变体与野生型及其他突变酶一样，以化学计量方式结合异柠檬酸。然而，与野生型酶不同的是，His(309)突变体不被金属异柠檬酸氧化裂解，这意味着金属离子未正常结合。由于野生型、H315Q和H319Q的圆二色光谱相似，这些氨基酸取代不会引起主要的构象变化。相比之下，His(309)的取代导致酶的CD光谱有可检测的变化，因此其二级结构也有变化。我们认为His(309)在辅酶结合中起重要作用，在有异丙醇存在时有助于二价金属离子的正确配位，并维持酶的正常构象。
