Pesce Alessandra, Milani Mario, Nardini Marco, Bolognesi Martino
Department of Physics CNR-INFM, and Center for Excellence in Biomedical Research, University of Genoa, Genoa, Italy.
Methods Enzymol. 2008;436:303-15. doi: 10.1016/S0076-6879(08)36017-0.
Protein matrix cavities and extended tunnels can effectively channel substrates and products to and from active sites in enzymes. Substrate and product channeling can enhance catalytic efficiency by reducing the intramolecular diffusion times to and from reaction centers. Moreover, protected transfer between sites may prevent the release of reactive intermediates, thus promoting efficiency or regulation in a series of interconnected reactions. This overview concerns the characterization of matrix tunnels in 2/2 hemoglobins, the recently described family of small hemoproteins (found in bacteria, plants, and unicellular eukaryotes) that form a separate cluster within the hemoglobin superfamily. Crystallographic investigations have shown that the 2/2 hemoglobin fold (a 2-on-2 alpha-helical sandwich) hosts a protein matrix tunnel system offering a potential path for ligand diffusion to and from the heme distal site. The tunnel topology is conserved in 2/2 hemoglobin group I, although with modulation of its size and/or structure. This article describes the methods that were adopted to characterize such matrix tunnels through analysis of the crystal structures and through binding of small apolar ligands to crystalline 2/2 hemoglobins. The methods are generally applicable and, in the case of 2/2 hemoglobins, underline the potential role of the tunnel system in supporting ligand diffusion to and from the heme, as well as ligand storage within the protein matrix.
蛋白质基质腔和延伸的通道能够有效地引导底物和产物进出酶的活性位点。底物和产物通道化可通过缩短底物进出反应中心的分子内扩散时间来提高催化效率。此外，位点之间的受保护转移可防止反应性中间体的释放，从而在一系列相互关联的反应中提高效率或促进调节。本综述关注2/2血红蛋白中基质通道的特征，2/2血红蛋白是最近描述的一类小的血红素蛋白家族（存在于细菌、植物和单细胞真核生物中），在血红蛋白超家族中形成一个单独的簇。晶体学研究表明，2/2血红蛋白折叠结构（一种2对2的α-螺旋三明治结构）包含一个蛋白质基质通道系统，为配体进出血红素远端位点提供了一条潜在路径。尽管通道的大小和/或结构有所调节，但通道拓扑结构在2/2血红蛋白I组中是保守的。本文描述了通过分析晶体结构以及使小的非极性配体与结晶的2/2血红蛋白结合来表征此类基质通道所采用的方法。这些方法具有普遍适用性，就2/2血红蛋白而言，突出了通道系统在支持配体进出血红素以及在蛋白质基质中储存配体方面的潜在作用。
