Zhao X, Vyas K, Nguyen B D, Rajarathnam K, La Mar G N, Li T, Phillips G N, Eich R F, Olson J S, Ling J
Department of Chemistry, University of California, Davis 95616, USA.
J Biol Chem. 1995 Sep 1;270(35):20763-74. doi: 10.1074/jbc.270.35.20763.
The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.
通过传统动力学技术、红外和共振拉曼光谱、1H NMR以及X射线晶体学，对大象肌红蛋白和抹香鲸肌红蛋白的L29F/H64Q突变体的功能、光谱和结构特性进行了详细比较。天然存在的大象蛋白与抹香鲸双突变体的特性之间存在显著对应关系，这两者均与天然抹香鲸肌红蛋白和单H64Q突变体的特性截然不同。这些结果以及Bisig等人最近的晶体结构测定结果（Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754 - 20762）证实，大象肌红蛋白第29位（B10）存在的是苯丙氨酸残基，而非已发表氨基酸序列中报道的亮氨酸残基。单个谷氨酰胺64（E7）取代使氧亲和力降低约5倍，并使自氧化速率提高3倍。在大象肌红蛋白和抹香鲸双突变体中，苯丙氨酸29（B10）的取代逆转了这些不利影响。后者这种基因工程蛋白最初被构建为具有适度低氧亲和力、大速率常数和增强的抗自氧化能力的血液替代原型。因此，我们基于配体结合和自氧化的提出机制合理设计的相同远端口袋组合在自然界中也被发现。
