JBC, Vol. 253, Issue 1, 252-256, Jan, 1978
Chemical structure of the active site of pig heart mitochondrial aspartate aminotransferase labeled with beta-chloro-l-alanine
Y. Morino and S. Tanase
Formate-induced inactivation of pig heart mitochondrial aspartate
aminotransferase by beta-chloro-L-alanine resulted in the modification of
the epsilon-amino group of the lysyl residue which is involved in the
formation of an aldimine bond with 4-formyl group of the coenzyme,
pyridoxal 5'-phosphate. The tryptic peptide isolated from the labeled site
of the enzyme was composed of 25 residues and exhibited positive circular
dichroism at 325 and 254 nm where the pyridoxyl chromophore of the labeled
site peptide absorbs, while the phosphopyridoxyl peptide isolated from the
boro-hydride-reduced enzyme did not show any ellipticity in this spectral
region. Its comparison with the analogous tryptic peptide from the labeled
site of the cytosolic isoenzyme revealed a high degree of homology in their
primary structures as well as in spectral properties. Structural analysis
of the labeled site peptide and mechanistic consideration of the labeling
process indicated that with both isoenzymes the phosphopyridoxyl group is
covalently bound to the alpha amino group of the alanyl moiety derived from
beta-chloro-L-alanine, the beta carbon of which is covalently linked to the
epsilon-amino group of the lysyl residue.
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