Urease activity in the crystalline state.
Urease activity in the crystalline state.
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结晶状态下的脲酶活性。
DOI：
10.1002/pro.5560041028
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Hausinger,RP
中科院分区：
文献类型：
--
作者：
Moncrief,MB;Hom,LG;Jabri,E;Karplus,PA;Hausinger,RP
在标准测定条件下，发现结晶产气克雷伯氏菌尿素酶的活性小于可溶性酶的0.05%。以2 M浓度存在于晶体储存缓冲液中的Li 2SO 4显示出通过混合抑制机制抑制可溶性尿素酶（游离酶的Ki为0.38 ± 0.05 M，酶-尿素复合物的Ki为0.13 ± 0.02 M）。然而，晶体的活性低于预期值的0.5%，这表明盐抑制并不能解释晶体活性几乎不存在的原因。晶体的溶解导致可溶性酶活性的约43%的恢复，这表明晶体生长期间的蛋白质变性不会导致催化速率的显著降低。最后，粉碎的晶体表现出只有三倍的活性增加，在完整的晶体，表明底物扩散到晶体的速率并没有显着限制酶的活性。我们的结论是，尿素酶是有效地在这种晶体形式的非活性，可能是由于构象限制与盖子覆盖的活性位点，并提出，少量的活性观察到的晶体表面或微量水平的酶溶解到晶体存储缓冲液中的有限的酶活性产生。
CrystallineKlebsiella aerogenesurease was found to have less than 0.05% of the activity observed for the soluble enzyme under standard assay conditions. Li2SO4, present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble urease by a mixed inhibition mechanism (Ki's of 0.38 ± 0.05 M for the free enzyme and 0.13 ± 0.02 M for the enzyme‐urea complex). However, the activity of crystals was less than 0.5% of the expected value, suggesting that salt inhibition does not account for the near absence of crystalline activity. Dissolution of crystals resulted in ˜43% recovery of the soluble enzyme activity, demonstrating that protein denaturation during crystal growth does not cause the dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a threefold increase in activity over that of intact crystals, indicating that the rate of substrate diffusion into the crystals does not significantly limit the enzyme activity. We conclude that urease is effectively inactive in this crystal form, possibly due to conformational restrictions associated with a lid covering the active site, and propose that the small amounts of activity observed arise from limited enzyme activity at the crystal surfaces or trace levels of enzyme dissolution into the crystal storage buffer.