Zou Jun, Zhang Rumin, Zhu Feng, Liu Jianjun, Madison Vincent, Umland Shelby P
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.
Biochemistry. 2005 Mar 22;44(11):4247-56. doi: 10.1021/bi0476230.
ADAM33 is an asthma susceptibility gene recently identified through a genetic study of asthmatic families [van Eerdewegh, et al. (2002) Nature 418, 426-430]. To understand the function of the gene product, the recombinant metalloproteinase domain of human ADAM33 was purified and tested for its substrate cleavage specificity using peptides derived from beta-amyloid precursor protein (APP). A single Ala substitution at the P2 position of a 10-residue APP peptide, YEVHHQKLVF, yielded a 20-fold more efficient substrate. Terminal truncation studies identified a minimal nine-residue core (P5-P4') important for ADAM33 recognition and cleavage. Full positional scanning of the 10-mer peptide using the 19 naturally occurring l-amino acids (excluding Cys) revealed a substrate specificity profile. A strong preference for Val or Ile at P3, Ala at P2, and Gln at P1' was observed. The substrate binding model based on the X-ray structure of the ADAM33-inhibitor complex supported the observed substrate specificity profile. On the basis of this, an improved substrate was designed and a fluorescence resonance energy transfer (FRET) assay was developed using a fluorogenic derivative of this substrate. Kinetic studies confirmed that the best substrate, FRET-P2 [K(Dabcyl)YRVAFQKLAE(Edans)K], was approximately 100-fold more efficient than the wild-type APP peptide substrate, with a k(cat)/K(m) value of (3.6 +/- 0.1) x 10(4) s(-)(1) M(-)(1). Using this substrate and the FRET assay, ADAM33 enzyme activity and thermal stability were characterized. ADAM33 dependence on buffer conditions, detergents, and temperature was examined, and optimal conditions were defined. Accurate K(i) values for tissue inhibitors of metalloproteinase and small molecule compounds were obtained.
ADAM33是一种哮喘易感基因，最近通过对哮喘家族的基因研究得以鉴定[范·埃尔德韦赫等人（2002年）《自然》418卷，426 - 430页]。为了解该基因产物的功能，对人ADAM33的重组金属蛋白酶结构域进行了纯化，并使用源自β - 淀粉样前体蛋白（APP）的肽段测试其底物切割特异性。在一个10个残基的APP肽YEVHHQKLVF的P2位置进行单个丙氨酸取代，得到了效率提高20倍的底物。末端截短研究确定了对ADAM33识别和切割至关重要的最小九个残基核心（P5 - P4'）。使用19种天然存在的L - 氨基酸（不包括半胱氨酸）对10聚体肽进行全位置扫描，揭示了底物特异性图谱。观察到在P3位置强烈偏好缬氨酸或异亮氨酸，在P2位置偏好丙氨酸，在P1'位置偏好谷氨酰胺。基于ADAM33 - 抑制剂复合物的X射线结构的底物结合模型支持了观察到的底物特异性图谱。据此，设计了一种改进的底物，并使用该底物的荧光衍生化试剂开发了一种荧光共振能量转移（FRET）测定法。动力学研究证实，最佳底物FRET - P2 [K（Dabcyl）YRVAFQKLAE（Edans）K]的效率比野生型APP肽底物高约100倍，k（cat）/K（m）值为（3.6 ± 0.1）×10⁴ s⁻¹ M⁻¹。使用该底物和FRET测定法，对ADAM33的酶活性和热稳定性进行了表征。研究了ADAM33对缓冲液条件、去污剂和温度的依赖性，并确定了最佳条件。获得了金属蛋白酶组织抑制剂和小分子化合物的准确K（i）值。
