Nugent P G, Albert A, Orprayoon P, Wilsher J, Pitts J E, Blundell T L, Dhanaraj V
Department of Crystallography, Birkbeck College, University of London, UK.
Protein Eng. 1996 Oct;9(10):885-93. doi: 10.1093/protein/9.10.885.
The loop exchange mutant chymosin 155-164 rhizopuspepsin was expressed in Trichoderma reesei and exported into the medium to yield a correctly folded and active product. The biochemical characterization and crystal structure determination at 2.5 A resolution confirm that the mutant enzyme adopts a native fold. However, the conformation of the mutated loop is unlike that in native rhizopuspepsin and involves the chelation of a water molecule in the loop. Kinetic analysis using two synthetic peptide substrates (six and 15 residues long) and the natural substrate, milk, revealed a reduction in the activity of the mutant enzyme with respect to the native when acting on both the long peptide substrate and milk. This may be a consequence of the different charge distribution of the mutated loop, its increased size and/or its different conformation.
环交换突变凝乳酶155 - 164根霉胃蛋白酶在里氏木霉中表达并分泌到培养基中，产生了正确折叠且有活性的产物。生化特性表征以及2.5埃分辨率的晶体结构测定证实该突变酶具有天然折叠结构。然而，突变环的构象与天然根霉胃蛋白酶不同，且涉及环中一个水分子的螯合。使用两种合成肽底物（分别为6个和15个残基长）以及天然底物牛奶进行的动力学分析表明，与天然酶相比，突变酶在作用于长肽底物和牛奶时活性降低。这可能是突变环电荷分布不同、尺寸增大和/或构象不同所致。
