Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids.
1987; Elsevier BV; Volume: 262; Issue: 17 Linguagem: Inglês
10.1016/s0021-9258(18)47543-6
ISSN1083-351X
AutoresE. W. Melzer, Hanns‐Ludwig Schmidt,
Tópico(s)Diet and metabolism studies
ResumoA method has been developed for the positional "C isotope analysis of pyruvate and acetate by stepwise quantitative degradation.On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined.The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 f 0.0007 and 1.0213 f 0.0017, respectively, and, with the enzyme from s. cerevisiae, the values are 1.0238 f 0.0013 and 1.0254 f 0.0016, respectively.A secondary isotope effect of 1.0031 f 0.0009 on C-3 (CH3group) was found with both enzymes.The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence.Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step ( k3) and the equilibrium isotope effect on the reversible substrate binding (kl, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope
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