Opposite effects of Ca2+ and GTP binding on tissue transglutaminase tertiary structure
Opposite effects of Ca2+ and GTP binding on tissue transglutaminase tertiary structure
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DOI：
10.1074/jbc.275.6.3915
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发表时间：
2000-02-11
影响因子：
4.8
通讯作者：
Mei, G
中科院分区：
文献类型：
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作者：
Di Venere, A;Rossi, A;Mei, G
组织转氨酶（tTG）属于催化蛋白质或肽之间的交联反应的一类酶。已知蛋白质活性通过Ca 2+和GTP结合而微调。在这项研究中，我们报告了这些配体对酶结构的影响，正如圆二色谱，稳态和动态荧光测量所揭示的那样，我们发现钙和GTP在蛋白质三级结构水平上诱导相反的构象变化，特别是金属离子负责蛋白质分子的小加宽，如各向异性衰减测量和疏水探针如1-苯胺基-8-萘磺酸（ANS）的结合所示，与Ca 2+不同，核苷酸结合增加了蛋白质动力学，将其旋转相关寿命从32 ns降低到25 ns，还防止ANS结合到蛋白质基质中。tTG的盐酸胍解折叠产生了一个三态变性机制，涉及一个中间物种与所谓的“熔融球”状态的特征。GTP结合（但不是Ca 2+）的效果对组织转氨酶的稳定性有重要影响，增加了从天然到中间物种的自由能变化至少接近0.7千卡/摩尔。此外，在GTP的存在下，获得了更大的稳定性的tTG高静水压力，这些研究结果表明，由GTP抑制tTG活性的分子机制基本上是由于蛋白质构象的变化，降低了蛋白质基质的溶剂的可及性，使得更难以暴露的活性位点。
Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides, The protein activity is known to be finely tuned by Ca2+ and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements, We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure, In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS), Unlike Ca2+, the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca2+) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximate to 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP, These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.