Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations1: in vivo kinetic characterization of 2,3-bisphosphoglycerate synthase/phosphatase using 13C and 31P NMR

Abstract

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%.

Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

“…The decrease in 2,3-BPG was caused by the reversed flux of the 2,3-BPG shunt after 14 days of storage (Figure 3A). Owing to the co-operative inhibition effects of 2,3-BPG synthase activity by protons, the reversed flux was speculated to be due to the decline in pH [19]. This reversed flux of the 2,3-BPG shunt is crucial in maintaining the activities of the latter part of glycolysis and the production of ATP in the latter half of the storage period (Figure 3B, bottom panel ).…”

Effects of Storage Time on Glycolysis in Donated Human Blood Units

“…We surveyed the literature and selected kinetic formulations that include known regulatory signals [32,33,34,35,36,37] (Section 2 in Supplementary Material). It is reasonable to expect that these regulatory signals are also present during refrigerated storage.…”

NMR spectroscopy in the study of carbohydrates: Characterizing the structural complexity

“…With appropriate calibration to compensate for the effects of differential relaxation and NOEs, time courses of 1-D 13 C NMR spectra enable the monitoring of the fluxes of key intermediates in the glycolytic pathway (88). Space limitations preclude their detailed consideration here but the following examples provide an indication of applications of 1-and 2-D techniques.…”