Williams D H, Murray E J
Roche Research Centre, Welwyn Garden City, Herts, UK.
FEBS Lett. 1994 Nov 14;354(3):267-70. doi: 10.1016/0014-5793(94)01136-2.
We have previously reported the crystal structure of truncated human collagenase (domain II) complexed with a low molecular weight inhibitor. Attempts to crystallize full-length active collagenase (i.e. domain II + III) have been hindered by autoproteolysis at the domain II/III junction at high protein concentrations. To overcome this problem, we have generated an inactive enzyme via a H149-->L,D151-->N double substitution which displaces the non-catalytic zinc atom, and shown that the altered collagenase is unable to cleave a synthetic substrate. We have also generated an 1251-->S substitution at the domain II/III junction and demonstrate an increased resistance to proteolysis compared to wild-type collagenase.
我们之前报道过截短的人胶原酶（结构域II）与一种低分子量抑制剂复合的晶体结构。在高蛋白浓度下，由于结构域II/III连接处的自蛋白水解作用，全长活性胶原酶（即结构域II + III）的结晶尝试受到了阻碍。为了克服这个问题，我们通过H149→L、D151→N双取代产生了一种无活性的酶，该取代使非催化锌原子移位，并表明改变后的胶原酶无法切割合成底物。我们还在结构域II/III连接处产生了I251→S取代，并证明与野生型胶原酶相比，其对蛋白水解的抗性增强。
