STRUCTURAL-ANALYSIS OF ZINC SUBSTITUTIONS IN THE ACTIVE-SITE OF THERMOLYSIN
STRUCTURAL-ANALYSIS OF ZINC SUBSTITUTIONS IN THE ACTIVE-SITE OF THERMOLYSIN
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DOI：
10.1002/pro.5560041001
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发表时间：
1995-10-01
期刊：
影响因子：
8
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
--
作者：
HOLLAND, DR;HAUSRATH, AC;MATTHEWS, BW
天然嗜热菌蛋白酶结合由三个蛋白质配体和一个水分子四面体配位的单个催化必需锌离子。在催化过程中，锌连接被认为从四倍变为五倍。用Cd 2+、Mn 2+、Fe 2+和Co 2+取代活性位点锌改变了催化活性（Holmquist B，Vallee BL，1974，J Biol Chem 249：4601-4607）。过量的锌会抑制这种酶。为了研究这些活性变化的结构基础，我们已经在1.7-1.9埃的分辨率下测定了一系列金属取代的嗜热菌蛋白酶的结构。取代的酶显示与野生型非常相似，除了两个溶剂分子在被认为被水合易断裂肽的两个氧占据的位置处与金属配位在过渡状态。因此，相对于锌取代的酶，钴取代的酶对某些底物的活性增强可能是由于过渡态的稳定性增强。Zn 2+和Co2+在米氏络合物中接受四面体配位的能力，以及在过渡态中的五重配位，也可能有助于它们在催化中的有效性。Cd 2 +-和Mn 2 +-取代的嗜热菌蛋白酶显示构象变化，破坏活性位点在不同程度上，可以解释相关的活动减少。构象变化不仅涉及必需的催化残基，Glu 143，但也协调的侧链旋转在相邻的残基Met 120和Leu 144。这些侧链运动中的一些类似于先前已经观察到的与“铰链-弯曲”运动相关的调节，所述“铰链-弯曲”运动被假定为在锌内切蛋白酶催化期间发生。在过量锌的存在下，观察到第二个锌离子在His 231处结合，在与天然嗜热菌蛋白酶结合的锌的3.2埃内，解释了抑制作用。
Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 Angstrom resolution.The structure of the Co2+-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+- and Mn2+-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these sidechain movements are similar to adjustments that have been observed previously in association with the ''hinge-bending'' motion that is presumed to occur during catalysis by the zinc endoproteases.In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 Angstrom of the zinc bound to native thermolysin, explaining the inhibitory effect.