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

摘要

pThis is the first in a series of three papers [see also Mulquiney and Kuchel (1999) Biochem. J. b342/b, 579-594; Mulquiney and Kuchel (1999) Biochem. J. b342/b, 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 iin iv/iiiv/io/i 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 iin iv/iitro/i is markedly different from that iin iv/iiiv/io/i. sup13/supC and sup31/supP NMR were used to monitor the time courses of selected metabolites when erythrocytes were incubated with or without [U-sup13/supC]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 kineticallyiin iv/iiiv/io/i. 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 iin iv/iiiv/io/i than iin iv/iitro/i; (3) the iK/isubm/sub of BPGS/P for 2,3-BPG is significantly higher than that measured iin iv/iitro/i; (4) the maximal activity of the phosphatase iin iv/iiiv/io/i is approximately twice that iin iv/iitro/i, when Psubi/sub is the sole activator (second substrate); and (5) 2-phosphoglycollate appears to play no role in the activation of the phosphatase iin iv/iiiv/io/i. Using the newly determined kinetic parameters, the percentage of glycolytic carbon flux that passes through the 2,3-BPG shunt in the normal iin iv/iiiv/io/i steady state was estimated to be 19%./p