Churchich J E, Wu C
J Biol Chem. 1982 Oct 25;257(20):12136-40.
N-Dansyl-2-oxopyrrolidine, a competitive inhibitor with respect to ATP, was used as a probe of the nucleotide binding site of pyridoxal kinase. It binds to an hydrophobic region of the catalytic site with a KD = 6 microM. Time emission anisotropy measurements yielded a rotational correlation time of 38 ns for the bound inhibitor. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the nucleotide binding site. Protein fluorescence quenching was used to determine the dissociation constants of the diastereomers of ATP beta S. Both diastereomers bind with a dissociation constant KD = 25 microM. The kinetic parameters Km and Vmax for the pyridoxal kinase reaction were determined for ATP and the diastereomers of ATP beta S in the presence of Mg(II), Co(II), Zn(II), and Cd(II). With Mg(II), pyridoxal kinase exhibits stereoselectivity for the A diastereomer of ATP beta S, Vmax ratio, A/B = 30. In the presence of Cd(II), the stereoselectivity is reversed and the B diastereomer of ATP beta S is the preferred substrate. As the divalent cations were varied in the series Mg(II), Co(II), Zn(II), and Cd(II), the A/B ratio was progressively lowered to the value of 0.2 found for Cd(II). These data indicate that the divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrates. The results obtained with the diastereomers of ATP alpha S suggest that pyridoxal kinase uses the divalent cation, delta,beta,gamma-bidentate nucleotide chelate as substrate.
N-丹磺酰-2-氧代吡咯烷是一种对ATP具有竞争性的抑制剂，被用作吡哆醛激酶核苷酸结合位点的探针。它以KD = 6微摩尔的解离常数与催化位点的疏水区域结合。时间发射各向异性测量得出结合抑制剂的旋转相关时间为38纳秒。N-丹磺酰-2-氧代吡咯烷通过与核苷酸结合位点的强相互作用而固定。蛋白质荧光猝灭用于测定ATPβS非对映体的解离常数。两种非对映体均以解离常数KD = 25微摩尔结合。在Mg(II)、Co(II)、Zn(II)和Cd(II)存在的情况下，测定了吡哆醛激酶反应对ATP以及ATPβS非对映体的动力学参数Km和Vmax。对于Mg(II)，吡哆醛激酶对ATPβS的A非对映体表现出立体选择性，Vmax比值A/B = 30。在Cd(II)存在的情况下，立体选择性反转，ATPβS的B非对映体是首选底物。随着二价阳离子在Mg(II)、Co(II)、Zn(II)和Cd(II)系列中变化，A/B比值逐渐降低至Cd(II)时发现的0.2值。这些数据表明二价阳离子与核苷三磷酸底物的β-磷酸基团配位。用ATPαS非对映体获得的结果表明，吡哆醛激酶使用二价阳离子δ,β,γ-双齿核苷酸螯合物作为底物。
