He M M, Voss J, Hubbell W L, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Biochemistry. 1995 Dec 5;34(48):15667-70. doi: 10.1021/bi00048a010.
Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269 --> His) and X (His322) [Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272] and helices VII (Asp237 --> His) and XI (Lys358 --> His) [He, M. M., Voss, J., Hubbell, W. L., & Kaback, H.R. (1995) Biochemistry 34, 00000--00000]. In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) [Jung, K., Jung, H., Wu, J., Prive, G. G., l& Kaback, H. R. (1993) Biochemistry 32, 12273]. Thus, mutants Arg302 --> His, Glu325 --> His, and Arg302 --> His/Glu325 --> His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance. Mutant Arg302 --> His binds Mn2+ with a KD of about 24 microM and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322. Mutant Arg302 --> His/Glu325 --> His also binds Mn2+ with a 1:1 stoichiometry, but the KD is decreased to about 13 microM. The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302 --> His/Glu325 --> His. In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding. The results provide strong support for the helix packing model proposed.
通过引入双组氨酸残基，将二价金属结合位点引入大肠杆菌乳糖通透酶中，已被用于证实螺旋VIII（Glu269→His）和螺旋X（His322）[Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272]以及螺旋VII（Asp237→His）和螺旋XI（Lys358→His）[He, M. M., Voss, J., Hubbell, W. L., & Kaback, H.R. (1995) Biochemistry 34, 00000--00000]之间的接近程度。在本文中，该方法用于证实并扩展螺旋IX（Arg302）与螺旋X（His322和Glu325）之间的关系[Jung, K., Jung, H., Wu, J., Prive, G. G., l& Kaback, H. R. (1993) Biochemistry 32, 12273]。因此，构建了突变体Arg302→His、Glu325→His以及Arg302→His/Glu325→His，并通过电子顺磁共振测定Mn2+结合情况。突变体Arg302→His以约24μM的解离常数（KD）结合Mn2+，化学计量比几乎为1，这很可能是因为302位的组氨酸残基与322位的天然组氨酸残基形成了一个金属结合位点。突变体Arg302→His/Glu325→His也以1:1的化学计量比结合Mn2+，但KD降至约13μM。结果表明，在突变体Arg302→His/Glu325→His中，Arg302与Glu325和His322都足够接近，从而形成了一个三齿金属结合位点。相比之下，在通透酶中用组氨酸取代322位具有天然组氨酸残基的Glu325，并不会导致Mn2+结合。这些结果为所提出的螺旋堆积模型提供了有力支持。
