Schaertl S, Konrad M, Geeves M A
Max-Planck Institut fur biophysikalische Chemie, Abteilung Molekulare Genetik, D-37070 Gottingen, Germany.
J Biol Chem. 1998 Mar 6;273(10):5662-9. doi: 10.1074/jbc.273.10.5662.
Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of gamma-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 x 10(6) M-1 s-1. Dephosphorylation by NDPs was 2-3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5'-O-(thiotriphosphate) (ATPgammaS) and guanosine 5'-O-(thiotriphosphate) (GTPgammaS) to NDP, but at (1)/(1000) of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATPgammaS and GTPgammaS occurred with kmax of 2.8 and 1.35 s-1 and Kd of 145 and 36 muM respectively. For dethiophosphorylation by a range of NDPs, kmax was in the range of 5-30 s-1, whereas Kd varied between 0.16 and 3.3 mM. Guanine had the lowest Kd values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2'-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3'-ribose.
核苷二磷酸激酶（NDKs）通过乒乓机制，催化γ-磷酸基团从核苷三磷酸（NTPs）经由活性位点组氨酸转移至核苷二磷酸（NDPs）。我们利用NDK磷酸化时发生的内在色氨酸荧光变化，来测定一系列核苷酸和核苷酸类似物的磷酸化及去磷酸化速率。对于天然核苷酸，磷酸化和去磷酸化速率与核苷酸浓度呈线性相关，直至速率过快无法测量。天然NTPs磷酸化的二级速率常数在0.7至13×10⁶ M⁻¹ s⁻¹之间变化。NDPs的去磷酸化速度比相应的磷酸化反应快2至3倍，而dNDPs的去磷酸化速度比等效的NDPs慢3至4倍。在所有情况下，鸟嘌呤的二级速率常数最高，其次是腺嘌呤，胞嘧啶核苷酸的二级速率常数最低。NDK还催化硫代磷酸从腺苷5'-O-（硫代三磷酸）（ATPγS）和鸟苷5'-O-（硫代三磷酸）（GTPγS）转移至NDP，但速率仅为等效磷酰基转移速率的1/1000。在这种情况下，观察到的磷酸化和去磷酸化速率常数对核苷酸浓度呈双曲线依赖关系。ATPγS和GTPγS的硫代磷酸化反应的最大反应速率（kmax）分别为2.8和1.35 s⁻¹，解离常数（Kd）分别为145和36 μM。对于一系列NDPs的去硫代磷酸化反应，kmax在5至30 s⁻¹范围内，而Kd在0.16至3.3 mM之间变化。鸟嘌呤的Kd值最低，胞嘧啶的Kd值最高。这些数据与核苷酸的快速可逆结合随后是限速磷酰基转移相一致。硫代磷酸仅改变磷酰基转移步骤的速率，而这两个事件均受碱基影响。核糖2'-羟基的修饰影响较小，而3'-核糖的修饰使磷酰基转移的总体速率降低1000倍。
