JBC, Vol. 250, Issue 17, 6870-6874, Sep, 1975
The stereochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate-6-phosphate aldolase
H. P. Meloche, L. Mehler and J. M. Wurster
In a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P as
catalyzed by 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) of
Pseudomonas putida, C--C synthesis occurred appreciably faster than C--3H
bond breaking. Since tritium is present in tritiated pyruvate in tracer
amounts, this result showed hydrogen isotope discrimination in pyruvate
deprotonation and suggests enolpyruvate generation to be at least partially
rate-limiting in the condensation reaction. Consequently, in a condensation
reaction between [3-3H, 2H,H]pyruvate of known chirality and
D-glyceraldehyde-3-P, the newly synthesized C--C bond would be enriched for
at what was the C--H bond of chiral pyruvate, discriminating against the
C--2H and C--3H bonds. Additional studies showed that condensations between
(3S)-[3-3H, 2H,H]- or (3R)-[3-3H, 2H,H]pyruvate and D-glyceraldehyde-3-P
yielded predominantly (3S)- or (3R)-2-keto-3-deoxy[3-3H, 2H]gluconate-6-P,
respectively. By comparison with sterochemical models, it was concluded
that condensation occurred with retention of configuration at C-3. Thus in
the turnover of substrates as catalyzed by this enzyme, both the exchanging
proton from water and D-glyceraldehyde-3-P attack the same face of the
enzyme-bound pyruvyleneamine.
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