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

This is the first in a series of three papers [see also Mulquiney and Kuche l (1999) Biochem. J. 342, 579-594; Mulquiney and Kuchel (1999) Biochem; J. 342, 595-602] that present a detailed mathematical model of erythrocyte met abolism which explains the regulation and control of 2,3-bisphosphoglycerat e (2,3-BPG) metabolism. 2,3-BPG is a modulator of haemoglobin oxygen affini ty and hence plays an important role in blood oxygen transport and delivery . This paper presents an in vivo kinetic characterization of 2,3-BPG syntha se/phosphatase (BPGS/P), the enzyme that catalyses both the synthesis and d egradation of 2,3-BPG. Much previous work had indicated that the behaviour of this enzyme in vitro is markedly different from that in vivo. C-13 and P -31 NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-C-13]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 N M R-derived data and simulations were achieved, it was possible to characte rize BPGS/P kinetically in vivo. This work revealed that: (1) the pH-depend ence of the synthase activity results largely from a strong co-operative in hibition 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 th an that measured in vitro; (4) the maximal activity of the phosphatase in v ivo is approximately twice that in vitro, when P-1 is the sole activator (s econd substrate); and (5) 2-phosphoglycollate appears to play no role in th e activation of the phosphatase in vivo. Using the newly determined kinetic parameters, the percentage of glycolytic carbon flux that passes through t he 2,3-BPG shunt in the normal in vivo steady state was estimated to be 19% .