The inhibition of purified bovine adrenal tyrosine hydroxylase by several product and substrate analogues has been studied to probe the kinetic mechanism. Norepinephrine, dopamine, and methylcatechol are competitive inhibitors versus tetrahydropterins and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine is an uncompetitive inhibitor versus tetrahydropterins and a competitive inhibitor versus tyrosine. The K(i) value for 3-iodotyrosine depends on the tetrahydropterin used. These results are consistent with tetrahydropterin binding first to the free enzyme followed by binding of tyrosine. 5-Deaza-6-methyl-tetrahydropterin is a noncompetitive inhibitor versus tetrahydropterins and tyrosine. The effect of varying the concentration of tyrosine on the K(i) value for 5-deaza-6-methyltetrahydropterin is consistent with the binding of this inhibitor to both the free enzyme and to an enzyme-dihydroxyphenylalanine complex. Dihydroxyphenylalanine also is a noncompetitive inhibitor versus tetrahydropterins and tyrosine; the effect of changing the fixed substrate is consistent with the binding of this inhibitor to both the free enzyme and to the enzyme-tetrahydropterin complex. The effect of pH on the K(i) values was determined in order to measure the pK(a) values of amino acid residues involved in substrate binding. Tight binding of catechols requires that a group with a pK(a) value of 7.6 be deprotonated. Binding of 3-iodotyrosine involves two groups with pK(a) values of 7.5 and about 5.5, one of which must be protonated for binding. Binding of 5-deaza-6-methyltetrahydropterin requires that a group on the free enzyme with a pK(a) value of 6.1 be protonated. The K(i) value for dihydroxyphenylalanine is relatively insensitive to pH, but the inhibition pattern changes from noncompetitive to competitive above pH 7.5, consistent with the measured pK(a) values of binding to the free enzyme and to the enzyme-tetrahydropterin complex.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|State||Published - 1988|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology