Martin J R, Mulder F A, Karimi-Nejad Y, van der Zwan J, Mariani M, Schipper D, Boelens R
Bijvoet Center for Biomolecular Research Utrecht University Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Structure. 1997 Apr 15;5(4):521-32. doi: 10.1016/s0969-2126(97)00208-6.
Research on high-alkaline proteases, such as serine protease PB92, has been largely inspired by their industrial application as protein-degrading components of washing powders. Serine protease PB92 is a member of the subtilase family of enzymes, which has been extensively studied. These studies have included exhaustive protein engineering investigations and X-ray crystallography, in order to provide insight into the mechanism and specificity of enzyme catalysis. Distortions have been observed in the substrate-binding region of subtilisin crystal structures, due to crystal contacts. In addition, the structural variability in the substrate-binding region of subtilisins is often attributed to flexibility. It was hoped that the solution structure of this enzyme would provide further details about the conformation of this key region and give new insights into the functional properties of these enzymes.
The three-dimensional solution structure of the 269-residue (27 kDa) serine protease PB92 has been determined using distance and dihedral angle constraints derived from triple-resonance NMR data. The solution structure is represented by a family of 18 conformers which overlay onto the average structure with backbone and all-heavy-atom root mean square deviations (for the main body of the molecule) of 0.88 and 1.21 A, respectively. The family of structures contains a number of regions of relatively high conformational heterogeneity, including various segments that are involved in the formation of the substrate-binding site. The presence of flexibility within these segments has been established from NMR relaxation parameters and measurements of amide proton exchange rates.
The solution structure of the serine protease PB92 presents a well defined global fold which is rigid with the exception of a restricted number of sites. Among the limited number of residues involved in significant internal mobility are those of two pockets, termed S1 and S4, within the substrate-binding site. The presence of flexibility within the binding site supports the proposed induced fit mechanism of substrate binding.
对高碱性蛋白酶（如丝氨酸蛋白酶PB92）的研究很大程度上源于其作为洗衣粉中蛋白质降解成分的工业应用。丝氨酸蛋白酶PB92是枯草杆菌蛋白酶家族的成员，该家族已得到广泛研究。这些研究包括详尽的蛋白质工程研究和X射线晶体学研究，以便深入了解酶催化的机制和特异性。由于晶体接触，在枯草杆菌蛋白酶晶体结构的底物结合区域观察到了扭曲。此外，枯草杆菌蛋白酶底物结合区域的结构变异性通常归因于灵活性。人们希望这种酶的溶液结构能提供有关这个关键区域构象的更多细节，并为这些酶的功能特性提供新的见解。
利用源自三共振核磁共振数据的距离和二面角约束，确定了269个残基（27 kDa）的丝氨酸蛋白酶PB92的三维溶液结构。溶液结构由18个构象体组成的家族表示，这些构象体叠加在平均结构上，主链和所有重原子的均方根偏差（对于分子主体）分别为0.88 Å和1.21 Å。该结构家族包含一些构象异质性相对较高的区域，包括参与底物结合位点形成的各种片段。这些片段内的灵活性已通过核磁共振弛豫参数和酰胺质子交换率测量得到证实。
丝氨酸蛋白酶PB92的溶液结构呈现出一个定义明确的整体折叠，除了少数受限位点外是刚性的。参与显著内部运动的有限数量的残基中，有底物结合位点内两个称为S1和S4的口袋中的残基。结合位点内的灵活性支持了所提出的底物结合诱导契合机制。
