Mulquiney P J, Bubb W A, Kuchel P W
Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia.
Biochem J. 1999 Sep 15;342 Pt 3(Pt 3):567-80.
This is the first in a series of three papers [see also Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594; Mulquiney and Kuchel (1999) Biochem. J. 342, 595-602] that present a detailed mathematical model of erythrocyte metabolism which explains the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is a modulator of haemoglobin oxygen affinity and hence plays an important role in blood oxygen transport and delivery. This paper presents an in vivo kinetic characterization of 2,3-BPG synthase/phosphatase (BPGS/P), the enzyme that catalyses both the synthesis and degradation of 2,3-BPG. Much previous work had indicated that the behaviour of this enzyme in vitro is markedly different from that in vivo. (13)C and (31)P NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-(13)C]glucose. Simulations of the experimental time courses were then made. By iteratively changing the parameters of the BPGS/P part of the model until a good match between the NMR-derived data and simulations were achieved, it was possible to characterize BPGS/P kinetically in vivo. This work revealed that: (1) the pH-dependence of the synthase activity results largely from a strong co-operative inhibition of the synthase activity by protons; (2) 3-phosphoglycerate and 2-phosphoglycerate are much weaker inhibitors of 2,3-BPG phosphatase in vivo than in vitro; (3) the K(m) of BPGS/P for 2,3-BPG is significantly higher than that measured in vitro; (4) the maximal activity of the phosphatase in vivo is approximately twice that in vitro, when P(i) is the sole activator (second substrate); and (5) 2-phosphoglycollate appears to play no role in the activation of the phosphatase in vivo. Using the newly determined kinetic parameters, the percentage of glycolytic carbon flux that passes through the 2, 3-BPG shunt in the normal in vivo steady state was estimated to be 19%.
这是三篇系列论文中的第一篇[另见Mulquiney和Kuchel（1999年）《生物化学杂志》342卷，579 - 594页；Mulquiney和Kuchel（1999年）《生物化学杂志》342卷，595 - 602页]，这些论文提出了红细胞代谢的详细数学模型，该模型解释了2,3 - 二磷酸甘油酸（2,3 - BPG）代谢的调节与控制。2,3 - BPG是血红蛋白氧亲和力的调节剂，因此在血液氧运输和输送中起重要作用。本文介绍了2,3 - BPG合酶/磷酸酶（BPGS/P）的体内动力学特征，该酶催化2,3 - BPG的合成与降解。此前许多研究表明，该酶在体外的行为与体内明显不同。当红细胞与[U - (13)C]葡萄糖一起孵育或不孵育时，使用(13)C和(31)P核磁共振来监测选定代谢物的时间进程。然后对实验时间进程进行模拟。通过反复改变模型中BPGS/P部分的参数，直到核磁共振得出的数据与模拟结果达到良好匹配，就有可能在体内对BPGS/P进行动力学表征。这项研究揭示：（1）合酶活性的pH依赖性很大程度上源于质子对合酶活性的强烈协同抑制；（2）在体内，3 - 磷酸甘油酸和2 - 磷酸甘油酸对2,3 - BPG磷酸酶的抑制作用比在体外弱得多；（3）BPGS/P对2,3 - BPG的米氏常数（K(m)）显著高于体外测量值；（4）当无机磷酸（P(i)）是唯一激活剂（第二底物）时，体内磷酸酶的最大活性约为体外的两倍；（5）2 - 磷酸乙醇酸在体内似乎对磷酸酶的激活没有作用。利用新确定的动力学参数，估计在正常体内稳态下通过2,3 - BPG支路的糖酵解碳通量百分比为19%。
