Fisher C L, Hallewell R A, Roberts V A, Tainer J A, Getzoff E D
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Free Radic Res Commun. 1991;12-13 Pt 1:287-96. doi: 10.3109/10715769109145797.
A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu,Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu 132, Glu 133, Lys 136, Thr 137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.
要全面理解酶 - 底物相互作用，需要在原子分辨率下详细了解其结构基础。已采用耦合计算和计算机图形学方法对晶体学和生化数据进行分析，以表征铜锌超氧化物歧化酶（SOD）识别超氧阴离子底物的分子基础。对牛SOD结构与来自15个物种的SOD序列进行详细分析，得出了有关序列保守性的意义和分子基础的新结果。已为所有23个不变残基以及表现出功能等效性的其他残基指定了特定作用。序列不变性主要由形成活性位点立体化学的15个残基主导，支持超氧化物歧化的主要生物学功能。利用晶体结构和定点突变体的数据，我们正在测试活性位点通道中各个残基的作用，包括（人SOD中的）Glu 132、Glu 133、Lys 136、Thr 137和Arg 143。结合分子柔性的静电计算表明，铜离子上方活性位点通道内及上方的正静电势区域在分子运动过程中扫过空间，以增强有助于酶快速催化速率的促进扩散。
