JBC, Vol. 250, Issue 17, 6875-6879, Sep, 1975
The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila
H. P. Meloche and C. T. Monti
In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC
4.1.2.21) is induced by growth on galatose while
2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These
enzymes catalyze identical reactions except for the configuration fixed at
C-4 during the condensation reaction. It was found with each enzyme that in
a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the
respective condensation products were formed 8 to 10 times faster than
tritium was released to water. Since pyruvate deprotonation is obligatory
for condensation, the above result requires a hydrogen isotope effect in
enolpyruvate formation, which must be then at least partially rate limiting
for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and
(3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by
each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation
product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis
with retention of configuration at C-3. This shows that the active sites of
both enzymes are asymmetric since solutes can only approach a single face
of the bound pyruvyl enolate. In addition, the respective aldehyde specific
portions of the two active sites must have opposite chiralities, with
respect to each other, for correctly orienting the carbonyl faces of the
incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4
of the respective condensation products.
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