Seymour Sean L, Klinman Judith P
Department of Chemistry, University of California, Berkeley, California 94720-1460, USA.
Biochemistry. 2002 Jul 9;41(27):8747-58. doi: 10.1021/bi020054g.
An earlier investigation of the temperature dependencies of rates and kinetic isotope effects (KIEs) in glucose oxidase (GO) used variants that differed in the extent of glycosylation at the surface of the protein. Kohen et al. [Kohen, A., Jonsson, T., and Klinman, J. P. (1997) Biochemistry 36, 2603-2611] presented evidence that the KIE on the Arrhenius prefactor varied as a function of protein modification, concluding that the degree of hydrogen tunneling at the active site was dependent on changes in mass at the surface. We now examine GO proteins containing polyethylene glycol (PEG) at their surface and a more extensively glycosylated form of GO, to distinguish simple mass effects from other sources of altered catalytic behavior. One PEG variant was created by modifying deglycosylated GO with short PEG chains (average of 350 Da each), while another contained a smaller number of long PEG chains (average of 5000 Da each). The light (146 kDa) and heavy (211 kDa) PEG variants and the hyperglycosylated variant display isotope effects on the Arrhenius prefactor that are similar (A(D)/A(T) = 0.55-0.62), while the unperturbed wild-type GO (WT-GO) is found to have an A(D)/A(T) that is reassessed as being close to unity. It appears that any modification of the protein surface away from that of the wild type gives rise to altered behavior for hydrogen transfer in the active site. We have also compared the effect of enthalpies of activation on both k(cat)/K(M) and k(cat) for the variants, introducing a new method to extract the k(cat)/K(M) rate constant and enthalpy of activation for the tritiated substrate from competitive KIE experiments. We find similar trends in Delta H(++) for both competitive and noncompetitive parameters and a smaller trend in k(cat) than reported earlier. Correlations are observed between A(D)/A(T) and both the enthalpies of activation and the thermal melt temperatures (T(M)) of the GO isoforms. In addition to the present study, there are now a number of examples where a perturbation of enzyme structure away from that of the wild type causes the observed KIE to become more temperature-dependent. The implications of these findings are discussed in the context of hydrogen tunneling and the relationship of protein structure and dynamics to this process.
早期对葡萄糖氧化酶（GO）中速率和动力学同位素效应（KIEs）的温度依赖性进行的一项研究使用了在蛋白质表面糖基化程度不同的变体。科恩等人[科恩，A.，琼森，T.，和克林曼，J.P.（1997年）《生物化学》36卷，2603 - 2611页]提供的证据表明，阿仑尼乌斯前因子上的KIE随蛋白质修饰而变化，得出活性位点处氢隧穿程度取决于表面质量变化的结论。我们现在研究在其表面含有聚乙二醇（PEG）的GO蛋白以及一种糖基化程度更高的GO形式，以区分简单的质量效应与催化行为改变的其他来源。一种PEG变体是通过用短PEG链（每条平均350道尔顿）修饰去糖基化的GO而产生的，而另一种含有较少数量的长PEG链（每条平均5000道尔顿）。轻（146 kDa）和重（211 kDa）PEG变体以及高糖基化变体在阿仑尼乌斯前因子上显示出相似的同位素效应（A(D)/A(T) = 0.55 - 0.62），而未受干扰的野生型GO（WT - GO）被发现其A(D)/A(T)经重新评估接近1。似乎蛋白质表面相对于野生型的任何修饰都会导致活性位点处氢转移行为的改变。我们还比较了活化焓对变体的k(cat)/K(M)和k(cat)的影响，引入了一种从竞争性KIE实验中提取氚化底物的k(cat)/K(M)速率常数和活化焓的新方法。我们发现竞争性和非竞争性参数的ΔH(++)有相似趋势，且k(cat)的趋势比早期报道的小。观察到A(D)/A(T)与GO同工型的活化焓和热熔温度（T(M)）之间存在相关性。除了本研究之外，现在有许多例子表明，酶结构相对于野生型的扰动会导致观察到的KIE变得更依赖于温度。这些发现的意义在氢隧穿以及蛋白质结构和动力学与该过程的关系的背景下进行了讨论。
