Phenylalanine hydroxylase (PAH) exists as an equilibrium of dimers and tetramers. However, there is little information concerning the inter- or intra-molecular interactions required for enzyme quaternary structure. It is predicted that the formation of a PAH tetramer will require at least two points of contact per enzyme subunit. Sequence analysis has suggested the existence of a C-terminal domain with characteristics of a leucine zipper or a variant of this called a coiled-coil. By deletion of 24 amino acids from the C-terminus or conversion of leucine 448 to an alanine residue, we have shown that this putative leucine zipper/coiled-coil domain is involved in the assembly of an active enzyme tetramer from dimers. The removal of this C-terminal domain of PAH reduces enzyme activity but does not abolish it. Furthermore, we report that an alanine 447 to aspartate mutation associated with phenylketonuria may affect subunit assembly which suggests the formation of enzyme tetramers is physiologically relevant. Our analysis of subunit interactions in vivo, show that in the absence of the C-terminal coiled-coil domain, dimers can form and this is only possible when the N-terminal domain is present. This provides the first evidence that N-terminal domain is required for multimerisation. We propose that the N-terminal regulatory domain in conjunction with the C-terminal coiled-coil domain, mediates the formation of fully active enzyme tetramers.
|Number of pages||10|
|Journal||Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology|
|Publication status||Published - 17 Feb 1998|
- Multimerisation domain
- Phenylalanine hydroxylase