The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N‐terminal region of the enzyme

Abstract

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL 0 ). The use of TRANSIL beads has allowed the determination of apparent dissociation constants (K d ) for the binding of the enzyme to negatively charged bilayers (K d = 230^380 W WM, at pH 6.0^7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal K K-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but K K-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH. ß

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

“…The relative distribution of 14-3-3␥ and -between CGG and cytosol is also calculated (inset). Effect of Liposome Binding on TH Activity-In agreement with previous findings (29), the binding of phosphorylated hTH1 (Ser(P) 19 -hTH1) to liposomes reduced the activity of the phosphorylated sample by 30% (Table 4). This reduction in activity is similar to that obtained for the unphosphorylated sample (data not shown).…”

“…hTH1 and 14-3-3␥ as Peripheral Proteins-As it emerges from this and previous works (26,28,29), both hTH1 and 14-3-3␥ may be classified as peripheral proteins. Although mainly cytoplasmic, these proteins can bind to membranes.…”

“…The 43-residue N-terminal region of TH was used in this work to investigate determinants for binding of this functional domain to membranes and 14-3-3 proteins. Comparison of the K d /S 0.5 values for the interaction of full-length hTH1 with either 14-3-3 proteins (17,48) or membranes (29) with those obtained in this work with the TH-(1-43) and THp-(1-43) peptides suggests that the main determinants for the interactions are included in this N-terminal domain. The TH-derived peptides are thus valuable to reveal the binding specificities.…”

“…It has further been shown that TH has affinity for isolated chromaffin granule membranes and negatively charged phospholipid layers (28,29). The binding of TH to membranes involves the N-terminal region of the enzyme as seen with recombinant human TH isoform 1 (hTH1), where truncated forms of the enzyme lacking up to residues 33-49 do not bind to membranes (29). Thus, both the phosphorylation-dependent TH binding to 14-3-3 and the interaction of TH with membranes occur through the same N-terminal region around Ser 19 (Fig.…”

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…However, additional factors, such as the relative rates of phosphorylation and dephosphorylation, turnover rates of unphosphorylated and phosphorylated enzyme, and its subcellular distribution (Thorolfsson et al 2002) must also be taken into account. Our finding that the 14-3-3 protein BMH1 shows a preferential inhibition of Ser19 dephosphorylation illustrates a mechanism whereby the pattern of phosphorylation may change with time and perhaps explain the differences found between in vitro systems and isolated cells.…”