Evans J A, Deutscher M P
J Biol Chem. 1976 Nov 10;251(21):6646-52.
We have examined the cation requirements of rabbit liver tRNA nucleotidyltransferase. The enzyme had an absolute requirement fo a divalent cation which could be satisfied by Mg2+, Mn2+ or Co2+. In contrast to the Escherichia coli enzyme, we have found no evidence to implicate Zn2+ in the action of rabbit liver tRNA nucleotidyltransferase. We have also identified a second cation requirement which was satisfied by divalent or monovalent cation or polyamines. The polyamines, spermine and spermidine, were most effective leading to 3-fold higher rates of nucleotide incorporation than could be obtained at any concentration of the other cations. However, the polyamines were not an absolute requirement for enzyme activity. The polyamine stimulation of tRNA nucleotidyltransferase was not due to alteration of the pH or ionic strength of the reaction mixture or reactivation of denatured tRNA or enzyme. Polyamines also had no effect on the apparent Km values of the substrates. Spermine increased the specificity of the enzyme for tRNA substrates and also inhibited the reverse action. Our results suggest that polyamines may be the normal counterions for tRNA in vivo, and that they affect the rate-limiting step in tRNA nucleotidyltransferase catalysis.
我们研究了兔肝tRNA核苷酸转移酶对阳离子的需求。该酶绝对需要一种二价阳离子，Mg2+、Mn2+或Co2+均可满足这一需求。与大肠杆菌的酶不同，我们没有发现任何证据表明Zn2+参与兔肝tRNA核苷酸转移酶的作用。我们还确定了第二个阳离子需求，二价或一价阳离子或多胺均可满足这一需求。多胺精胺和亚精胺最为有效，与其他阳离子在任何浓度下所能达到的核苷酸掺入率相比，它们能使掺入率提高3倍。然而，多胺并非酶活性的绝对必需物。多胺对tRNA核苷酸转移酶的刺激作用并非由于反应混合物的pH值或离子强度的改变，也不是由于变性tRNA或酶的重新激活。多胺对底物的表观Km值也没有影响。精胺提高了该酶对tRNA底物的特异性，同时也抑制了反向反应。我们的结果表明，多胺可能是体内tRNA的正常抗衡离子，并且它们影响tRNA核苷酸转移酶催化作用中的限速步骤。
