Kiss András L, Palló Anna, Náray-Szabó Gábor, Harmat Veronika, Polgár László
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest 112, P.O. Box 7, Hungary.
J Struct Biol. 2008 May;162(2):312-23. doi: 10.1016/j.jsb.2008.01.012. Epub 2008 Feb 2.
It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme.
人们普遍认为，丝氨酸蛋白酶的催化活性主要取决于天冬氨酸-组氨酸-丝氨酸催化三联体以及该基序附近的其他残基。其中一些残基形成氧阴离子结合位点，通过与带负电荷的氧阴离子形成氢键来稳定四面体中间体。在嗜热古菌嗜热栖热菌的酰基氨基酸酰基肽酶中，Gly369的主链NH基团是形成氧阴离子结合位点的氢键供体之一。His367的侧链是所有物种酰基氨基酸酰基肽酶中的保守残基，它固定着包含Gly369的环。对H367A突变体晶体结构的测定表明，这个包括Gly369的环明显移动，这解释了观察到的特异性速率常数下降三个数量级的现象。对于野生型酶，ln(k(cat)/K(m))与1/T的关系偏离线性，这表明与酶-底物复合物分解为产物相比，随着温度升高，酶-底物复合物解离的速率增强更大。相比之下，H367A变体给出了线性的阿伦尼乌斯图，其反应与不利的活化熵相关。这些结果表明，一个距离活性位点相对较远的残基可以在不改变酶整体结构的情况下显著影响酰基氨基酸酰基肽酶的催化活性。
