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首页> 外文期刊>Biochemistry >UTILITY OF A NOVEL SPIN-LABELED NUCLEOTIDE IN INVESTIGATION OF THE SUBSTRATE AND EFFECTOR SITES OF PHOSPHORIBULOKINASE
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UTILITY OF A NOVEL SPIN-LABELED NUCLEOTIDE IN INVESTIGATION OF THE SUBSTRATE AND EFFECTOR SITES OF PHOSPHORIBULOKINASE

机译：新型旋转标记的核苷酸在研究磷酸珠蛋白激酶的底物和有效位点中的实用性

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摘要

The activated spin-label 3-(2-bromoacetamido)proxyl modifies the sulfur atom of phosphorothioate-containing AMP, ADP, and ATP analogs in a facile reaction that produces a new series of spin-labeled nucleotides. One of these products, adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate) (ATP gamma SAP), has been evaluated as a structural probe for Rhodobacter sphaeroides phosphoribulokinase (PRK). When incubated with affinity-purified enzyme that contains tightly bound substrate ATP, ATP gamma SAP binds noncooperatively to the allosteric site (n = 1; K-D = 8 mu M). Probe bound in this site is displaced (K-1/2 = 100 mu M) by the allosteric effector, NADH, at concentrations comparable to those required for enzyme activation (K-a 133 mu M). In the presence of NADH, when PRK's substrate site is vacant, ATP gamma SAP binds in a cooperative mode (Hill coefficient approximate to 2.9; K-D = 20 mu M). In the absence of NADH, ATP gamma SAP mimics ATP by exhibiting nonequilibrium binding to PRK. The observations with phosphoribulokinase, together with the straightforward nature of the methodology documented for synthesis and isolation of this class of spin-labeled nucleotides, suggest that these analogs have potentially wide application as structural probes.
机译：活化的自旋标记的3-（2-溴乙酰氨基）脯氨酰在易于产生新的自旋标记核苷酸系列的反应中修饰了含硫代磷酸酯的AMP，ADP和ATP类似物的硫原子。这些产品之一，腺苷5'-O-（S-乙酰氨基己基3-硫代三磷酸）（ATPγSAP），已被评估为球形球形红细菌磷酸核糖激酶（PRK）的结构探针。当与含有紧密结合的底物ATP的亲和纯化酶一起孵育时，ATPγSAP不合作地结合到变构位点（n = 1; K-D = 8μM）。结合在该位点的探针被变构效应物NADH置换（K-1 / 2 = 100μM），其浓度与酶激活所需的浓度（K-a 133μM）相当。在存在NADH的情况下，当PRK的底物位点空缺时，ATPγSAP以协作模式结合（希尔系数约为2.9; K-D = 20μM）。在没有NADH的情况下，ATPγSAP通过表现出与PRK的非平衡结合来模仿ATP。用磷酸核糖激酶的观察结果，以及为合成和分离这类自旋标记核苷酸而记录的方法的直接性质，表明这些类似物作为结构探针具有潜在的广泛应用。

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