Hummel C F, Gerber B R, Carty R P
Int J Pept Protein Res. 1984 Jul;24(1):1-13. doi: 10.1111/j.1399-3011.1984.tb00921.x.
4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group.
4-砷酸-2-硝基氟苯在牛胰核糖核酸酶A的阴离子结合位点发生选择性反应。主要衍生物是无活性的41-（4-砷酸-2-硝基苯基）核糖核酸酶A（产率45%）。其他产物是具有酶活性的1-α-（4-砷酸-2-硝基苯基）核糖核酸酶A（产率11%）和双取代的无活性1,41-双-（4-砷酸-2-硝基苯基）核糖核酸酶A（产率25%）。2'（3'）-O-溴乙酰尿苷仅与41-（4-砷酸-2-硝基苯基）核糖核酸酶A的组氨酸-12残基反应，反应速率约为未修饰酶观察到速率的四分之一。观察到饱和动力学，蛋白质-抑制剂复合物的解离常数为0.096±0.023 M。复合物分解的一级单分子分解常数为8.9±2.9×10⁻⁴ s⁻¹。2'-溴乙酰氨基-2'-脱氧尿苷与41-（4-砷酸-2-硝基苯基）核糖核酸酶A的反应速度比2'（3'）-O-溴乙酰尿苷慢25倍。溴乙酸与41-（4-砷酸-2-硝基苯基）核糖核酸酶A主要在组氨酸-119残基处反应，反应速率比未修饰酶低45倍。烷基化研究结果表明，二价阴离子砷酸酯不占据酶中的磷酸结合位点，但距离足够近，足以解释溴乙酸结合的减少以及组氨酸-12和-119亲核反应性的降低。所有这些效应都可以用砷酸硝基苯基基团对活性位点区域的局部静电扰动来解释。
