Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Abstract

The kinetic and equilibrium isotope effects on the fructose-1,6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13 C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13 C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13 (V max /K m ) on C-3 was 1.016 ؎ 0.007 and 0.997 ؎ 0.009 on position C-4, and the equilibrium isotope effects K 12 /K 13 on these positions were 1.0036 ؎ 0.0002 and 1.0049 ؎ 0.0001.The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13 C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13 C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13 C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.The relative enrichment of carbon-13 in the carboxyl group of amino acids, as observed by Abelson and Hoering (1) in 1961, was the first indication for the existence of non-statistical isotope distributions in biological compounds. Later results on acetic acid (2) and on acetoin (3) gave evidence that this observation was just one example of a common phenomenon. In order to find a general explanation for this observation Galimov (4) discussed that even in chemically unequilibrated systems, e.g. biological systems, a microscopic reversibility in enzymatic reactions is the origin for a thermodynamically ordered isotope distribution. The author's calculations could in fact explain some of the isotopic patterns of natural compounds known at that time. However, the presumption of a general thermodynamic equilibrium in biological systems is probably not realistic. In our opinion kinetic isotope effects on enzymatic reactions should be considered as primary causes for isotope discriminations. This has been proven for the primary CO 2 -fixing reactions (5-8). In secondary metabolism, the isotope effect on the pyruvate dehydrogenase reaction has been made responsible for the general depletion of 13 C in metabolites of acetyl-CoA, such as fatty acids or isoprenoids (9 -15).More detailed interpretations were possible when the total isotopic patterns of primary and secondary metabolites became available. Especially, our corresponding investigations on glucose indicated an enrichment of 13 C in positions C-3 and C-4, and a depletion of 13 C in positions C-1 and C-6 (Fig. 1) of this important primary metabolite (16). We have a...

Nonstatistical 13C Distribution during Carbon Transfer from Glucose to Ethanol during Fermentation Is Determined by the Catabolic Pathway Exploited

“…Thus, the C-3 G enters the CH 3 of ethanol, in marked contrast to the EMP pathway. Although no 13 C KIE studies have been done on the KDPG aldolase, its mechanism (27) is closely similar to that of fructose 1,6-bisphosphate aldolase (28), for which a normal KIE of 1.0254 has been determined (17). This value is in consensus with the app KIE CH 3 found here.…”

“…By calculation, the deduced KIE CH 3 for KDPG aldolase on the C-3 G is 1.0021. Although only 10% the magnitude of the primary KIE determined for fructose-1,6-bisphosphate aldolase (17), this once again indicates that reactions are occurring under conditions far from V max /K m . In addition, these two enzymes differ mechanistically (27).…”

“…Although no isotopic data are available for this reaction, its similarity to the better studied fructose-1,6-bisphosphate aldolase (35) for which a normal KIE of 1.0254 (17) has been determined, making it probable that the app KIE CH 3 ϭ 1.0057 is due to the activity of this enzyme. That this value is smaller than determined for the fructose-1,6-phosphate aldolase might reflect the sp 2 -sp 3 change during the alcohol dehydrogenase step, which is likely to manifest a small secondary inverse KIE (see below).…”

“…These mixtures of isotopomers are determined by the presence of isotope effects in the Calvin cycle and indicate the role of certain enzymes in the 13 C isotope discrimination. For example, the aldol condensation of glyceraldehyde-3-phosphate (GAP) with dihydroxyacetone phosphate by fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) to form fructose 1,6-bisphosphate could be identified as the origin of the relative 13 C enrichment at the C-3 and C-4 positions of glucose (17).…”

Diel variations in the carbon isotope composition of respired CO<sub>2</sub> and associated carbon sources: a review of dynamics and mechanisms

“…During the day, the synthesis of transitory starch is either under plant internal control to adapt the storage C supply to environmental conditions (Zeeman et al, 2007) or occurs mainly when the utilisation of newly produced triose-phosphates from the chloroplast becomes rate limiting to carbon assimilation (Beck and Ziegler, 1989). Gleixner et al (1998) suggested that transitory starch is 13 C enriched relative to soluble sugars because of the isotope effects on the "aldolase-reaction" determined originally Gleixner and Schmidt (1997). As a consequence, 13 Cdepleted triose phosphates are exported from the chloroplast, which are used for sucrose production during the light period and thus influence δ 13 C res .…”