Tralau C, Greller G, Pajatsch M, Boos W, Bohl E
Departments of Mathematics and Biology, University of Konstanz, Konstanz, D-78457, Germany.
J Theor Biol. 2000 Nov 7;207(1):1-14. doi: 10.1006/jtbi.2000.2140.
Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.
传统上，细菌转运系统被视为具有可饱和结合位点的酶，由此产生表观转运K(m)，而最大转运速率被认为等同于酶促反应的V(max)。因此，米氏理论通常应用于转运数据的分析，K(m)和V(max)通过处理在不同底物浓度下的转运速率所获得的数据得出。这种分析默认转运系统的底物识别位点可自由接触底物。然而，情况并非总是如此。在具有微摩尔范围内高亲和力的系统中，或那些识别大分子底物的系统中，或那些具有高V(max)的系统中，通过外膜的扩散可能成为限速步骤，特别是在外部底物浓度较低时。在这种情况下，总体转运速率（从培养基到细胞质）对培养基中底物浓度的依赖性将不再遵循米氏动力学。通过分析转运数据与相应理想米氏图的偏差，我们开发了一种方法，该方法可让我们确定通过外膜的扩散限制。该方法使我们能够找到即使在外膜存在强烈扩散限制的条件下在内膜发挥作用的转运系统的正确K(m)。该模型用大肠杆菌麦芽糖结合蛋白依赖性ABC转运体进行了测试和验证。大肠杆菌sn-甘油-3-磷酸的相应系统以及产酸克雷伯菌的α-环糊精转运系统用作测试系统。
