J. Biol. Chem., Vol. 261, Issue 16, 7105-7108, 06, 1986
Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase
HP Tobler, P Christen and H Gehring
In the course of a half-reaction of enzymic transamination, the aldimine
adduct formed between the coenzyme pyridoxal 5'-phosphate and the amino
acid substrate tautomerizes to the ketimine intermediate which is then
hydrolyzed to the oxo acid product and the pyridoxamine 5'-phosphate form
of the enzyme. In the reverse half-reaction the tautomerization is
initiated by the removal of a proton from the pro-S position at C-4' of the
PMP moiety of the ketimine intermediate. The present study investigates the
question whether the pro-S hydrogen at C- 4' of PMP is labilized by its
active site environment independently of the formation of the ketimine
intermediate, i.e. in the absence of substrate. Reconstitution of
apoaspartate aminotransferase (mitochondrial isoenzyme from chicken) with
[4'-3H] PMP results indeed in a stereospecific exchange of pro-S 3H with
solvent water. The exchange follows first order kinetics (t 1/2 = 23 min at
pH 7.5 and 25 degrees C). Unbound PMP showed no measurable exchange.
Rigorous control experiments excluded the possibility that the observed
exchange was due to a transamination reaction of the enzyme with
contaminating oxo acid substrates. The newly observed stereospecific
exchange reaction allows to investigate the acid/base properties of C-4'
and the modulating effects of its active site environment independently of
the preceding and following steps of enzymic transamination.
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