Philips A V, Robbins D J, Coleman M S, Barkely M D
Biochemistry. 1987 May 19;26(10):2893-903. doi: 10.1021/bi00384a034.
Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.
通过使用单克隆抗体亲和柱分离出人胸腺腺苷脱氨酶。这种快速、高效的方法所产生的高度纯化的酶分子量为44,000。利用小分子对蛋白质固有荧光的猝灭来探测该酶及酶 - 抑制剂复合物中色氨酸残基的可及性。人腺苷脱氨酶在295 nm激发下的荧光发射光谱在约335 nm处有最大值，量子产率为0.03。添加极性荧光猝灭剂碘化物和丙烯酰胺使峰向蓝光方向移动，而疏水性猝灭剂三氯乙醇使峰向红光方向移动，表明发射光谱是不均匀的。从这些猝灭剂获得的荧光猝灭参数表明蛋白质中的色氨酸环境相对疏水。基态和过渡态类似物抑制剂的结合均导致酶荧光强度降低，这表明一个或多个色氨酸可能靠近活性位点。荧光强度降低的动力学与过渡态抑制剂复合物中缓慢的构象改变一致。还对酶 - 抑制剂复合物进行了使用极性和非极性猝灭剂的荧光猝灭实验。所有酶 - 抑制剂复合物的猝灭参数与未复合酶的不同，表明抑制剂结合会导致腺苷脱氨酶构象发生变化。为作比较，在有无抑制剂的情况下对小牛腺苷脱氨酶进行了平行猝灭研究。虽然来自这两种来源的腺苷脱氨酶之间明显存在显著的结构差异，但我们的数据表明两种酶在结合基态和过渡态抑制剂时都会发生构象变化。
