The carboxy‐terminal peptide of detyrosinated α tubulin provides a minimal system to study the substrate specificity of tubulin–tyrosine ligase

The ATP‐dependent tubulin–tyrosine ligase (TTL) restores the carboxy‐terminal tyrosine of α tubulin in αβ tubulin that has been previously detyrosinated. Here we show that the carboxy‐terminal tetradecapeptide of detyrosinated α tubulin is used by TTL as a substrate, albeit at 50‐fold lower efficiency than αβ tubulin. The minimal system provided by the TTL/peptide combination mirrors the TTL/tubulin system in all aspects tested, and shows a pronounced substrate inhibition. Synthetic peptides varying in length and/or containing single amino acid replacements were used to analyze the TTL specificity for the carboxy‐terminal sequence of detyrosinated α tubulin. Peptides ending like α tubulin with the sequence Gly‐Glu‐Glu are optimally tyrosinated once a peptide length of 12 residues is reached. Position ‐1 of this recognition sequence, to which the tyrosine is added, must be glutamic acid. Position ‐2 accepts only an acidic amino acid but glutamic acid is by far preferred over aspartic acid. These results explain why a subpopulation of brain α tubulin, which ends with the sequence Gly‐Glu, is not tyrosinated by TTL. The carboxy‐terminal dodecapeptide of brain α tubulin with its polyglutamyl side‐chain on position ‐6 shows the same substrate activity as the corresponding synthetic peptide lacking the side‐chain. We discuss the substrate specificity of TTL for different α tubulins and speculate why tubulin is a better substrate than the optimal peptide covering the carboxy‐terminal of detyrosinated α tubulin.