Labeling of the active site of cytoplasmic aspartate aminotransferase by β-chloro-L-alanine

  • Morino Y
  • Okamoto M
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Abstract

Syncatalytic inactivation of pig heart cytoplasmic aspartate aminotransferase by β-chloro-[U-14C]L-alanine resulted in the incorporation of radioactivity corresponding to one mole of the label per mole of the monomeric unit of the enzyme. A borohydride-reduced and then carboxymethylated preparation of the labeled enzyme was digested by trypsin. A radioactive peptide was isolated and found to contain a covalently linked pyridoxyl derivative which absorbed at 325 nm. The amino acid sequence of this peptide was Tyr-Phe-Val-Ser-Glu-Gly-Phe -Glu-Leu-Phe-Cys-Ala-Gln-Ser-Phe-Ser-Lys{black star}-Asn-Phe-Gly-Leu-Tyr-Asn-Glu-Arg. In the peptide the phosphopyridoxyl group seems to be covalently bound via alanyl moiety derived from β-chloro-L-alanine, the β-carbon atom of which is covalently linked to the ε{lunate}-nitrogen atom of the lysyl residue(Lys{black star}). From a comparison with the amino acid composition of the phosphopyridoxyl peptide isolated from the tryptic digest of a borohydride-reduced holoenzyme, it was concluded that the modified lysul residue was identical to that involved in binding pyridoxal phosphate to the apoenzyme. © 1973.

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Authors

  • Yoshimasa Morino

  • Mitsuhiro Okamoto

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