Moncrief M B, Hom L G, Jabri E, Karplus P A, Hausinger R P
Department of Biochemistry, Michigan State University, East Lansing 48824-1101, USA.
Protein Sci. 1995 Oct;4(10):2234-6. doi: 10.1002/pro.5560041028.
Crystalline Klebsiella aerogenes urease 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 approximately 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 three-fold 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.
在标准测定条件下，发现结晶型产气克雷伯氏菌脲酶的活性不到可溶性酶活性的0.05%。晶体储存缓冲液中浓度为2 M的硫酸锂通过混合抑制机制抑制可溶性脲酶（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，晶体的活性不到预期值的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。晶体溶解后，可溶性酶活性的回收率约为43%，这表明晶体生长过程中的蛋白质变性不会导致催化速率大幅降低。最后，碾碎的晶体活性仅比完整晶体高两倍，这表明底物扩散到晶体中的速率不会显著限制酶活性。我们得出结论，脲酶在这种晶体形式下实际上是无活性的，可能是由于与覆盖活性位点的盖子相关的构象限制，并提出观察到的少量活性源于晶体表面有限的酶活性或微量的酶溶解到晶体储存缓冲液中。
