Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Abstract

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.

Scheme 1

Fig. 1

N-Terminal amino acid sequences of human tyrosine hydroxylase isoforms and rat tyrosine hydroxylase. The remaining sequences are identical among the human isoforms. The phosphorylated residues in the rat enzyme are in bold.

Fig. 2

Concentration dependence of DOPA binding to hTyrH₁ (filled circles) and hTyrH₄ (open circles). The conditions were as described for Table 2.

Fig. 3

Spectral changes during displacement by DHN of DOPA (a) or dopamine (b and c) from unphosphorylated (a and b) and phosphorylated (c) hTyrH_4. Enzyme (30 μM) was incubated with 60 μM L-DOPA or dopamine for 15 min in 10% glycerol, 100 mM KCl, 0.2 mM DTPA, 50 mM HEPES, pH 7, at 10°C, before the spectra indicated by solid lines were taken. DHN was then added to give a final concentration of 1 mM; the spectra indicated by dashed lines were taken after 2.8 h (a) or 12.5 h (b and c).

Fig. 4

Effects of phosphorylation (solid bars) on the rate constants for association (a) and dissociation (b) of DOPA from TyrH isoforms. The conditions were as described for Fig. 3.