Probing the role of highly conserved residues in triosephosphate isomerase – analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme
Probing the role of highly conserved residues in triosephosphate isomerase – analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme
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DOI：
10.1111/febs.13384
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
D. Bandyopadhyay;M. Murthy;H. Balaram;P. Balaram
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文献类型：
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作者：
D. Bandyopadhyay;M. Murthy;H. Balaram;P. Balaram
酶中高度保守的残基经常聚集在活性位点附近，这表明功能限制决定了这些位点上氨基酸残基的性质。以恶性疟原虫磷酸三糖异构酶(PfTIM)酶(EC 5.3.1.1)为模板，研究了与质子转移循环无关的和75位点突变的影响。苏氨酸(T)在第75位是完全保守的，而在位只有谷氨酰胺(Q)和谷氨酸(E)。报道了四个T75(T75S/V/C/N)和两个Q64(Q64N/E)突变体的生物物理和动力学数据。在Q64E和Q64N突变体中，二聚体结构被削弱，而在所有四个T75突变体中，二聚体的完整性没有受到损害。通过测量酶活性的浓度依赖关系，可以估计二聚体解离的Kd值(Q64N=73.7±9.2 nm，Q64E=44.6±8.4 nm)。T75S/V/C突变体的活性与野生型酶相当，而T75N的活性下降了四倍。所有四个T75突变体的活性都在35°C到45°C之间急剧下降。T75S/V/N突变体的晶体结构测定提供了对局部相互作用变化的洞察，其中T75N突变体显示的变化最大。氢键相互作用决定了二聚体的稳定性，限制了位置上的残基选择为谷氨酰胺(Q)和谷氨酸(E)。在第75位，对Thr(T)的压倒性偏好可能是由维持酶活性的温度稳定性的必要性决定的。
Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild‐type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 °C and 45 °C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen‐bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity.