Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase.
The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.
Deans
N L
NL
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610-0245.
Allison
R D
RD
Purich
D L
DL
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D007980:Levodopa / Q000494:pharmacology; D008962:Models, Theoretical; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism
D013439:Sulfhydryl Reagents; D014404:Tubulin; D000249:Adenosine Monophosphate; D007980:Levodopa; D000244:Adenosine Diphosphate; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase; D005033:Ethylmaleimide