The carboxy-terminal peptide of detyrosinated alpha 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 alpha tubulin in alpha beta tubulin that has been previously detyrosinated. Here we show that the carboxy-terminal tetradecapeptide of detyrosinated alpha tubulin is used by TTL as a substrate, albeit at 50-fold lower efficiency than alpha beta 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 alpha tubulin. Peptides ending like alpha 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 alpha tubulin, which ends with the sequence Gly-Glu, is not tyrosinated by TTL. The carboxy-terminal dodecapeptide of brain alpha 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 alpha tubulins and speculate why tubulin is a better substrate than the optimal peptide covering the carboxy-terminal of detyrosinated alpha tubulin.
Rüdiger
M
M
Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Germany.
Wehland
J
J
Weber
K
K
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008868:Microtubule Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine
D008868:Microtubule Proteins; D009842:Oligopeptides; D014404:Tubulin; D011099:Polyglutamic Acid; D014443:Tyrosine; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase