Ingram Richard N, Orth Peter, Strickland Corey L, Le Hung V, Madison Vincent, Beyer Brian M
Department of Structural Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Protein Eng Des Sel. 2006 Apr;19(4):155-61. doi: 10.1093/protein/gzj014. Epub 2006 Feb 3.
The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.
肿瘤坏死因子-α转化酶（TACE）的结晶对于理解新化学实体的构效关系很有帮助。然而，TACE发生自蛋白水解的倾向使得酶的处理变得困难，并阻碍了可用于抑制剂浸泡的晶体形式的鉴定。发现TACE的自蛋白水解具有特异性（Y352-V353），且发生在靠近活性位点P侧的一个柔性环内。自蛋白水解速率与TACE浓度成正比，表明是双分子反应机制。使用替代肽对S(1)'亚位点进行了有限的特异性研究，并提出了能稳定Y352-V353位置环的蛋白水解的取代方案。生成了两种突变蛋白酶（V353G和V353S），并证明它们对自蛋白水解具有高度抗性。比较了抗性更强的突变体（V353G）和野生型TACE的动力学，结果表明一组竞争性抑制剂的IC(50)值几乎相同。然而，突变体对更大底物（P6 - P(6)'）的k(cat)/K(m)比野生型酶低约5倍。野生型和突变体复合物结构的比较表明，外围P侧环（包含突变位点）有细微位移，这可能与底物结合/周转有关，也可能解释了轻微的动力学差异。这种稳定形式的TACE的表征产生了一种具有类似天然动力学性质的酶，并鉴定出一种适合抑制剂浸泡和结构测定的新晶体形式。
