Conjugation of phenylalanine hydroxylase with polyubiquitin chains catalysed by rat liver enzymes

Phenylalanine hydroxylase (PAH, EC 1.14.16.1) is a highly regulated liver enzyme which catalyses the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the catabolic pathway of this amino acid. Among the approx. 400 different mutations of human (h) PAH, frequently associated with the metabolic disease phenylketonuria. a low stability is a characteristic property when expressed in eucaryotic cells. In this study, the pathway of hPAH degradation is addressed with focus on its conjugation with polyubiquitin chains catalysed by the ubiquitin-conjugating enzyme system (E1, E2, E3) isolated from rat liver by covalent affinity chromatography on ubiquitin-Sepharose In the reconstituted in vitro ubiquitination assay, the enzyme system catalysed both the formation of free polyubiquitin chains and the polyubiquitination of wild-type (wt) hPAH and its 'catalytic domain' (Delta N102/Delta C24-hPAH) as visualized by two-dimensional electrophoresis. The ubiquitination of wt-PAM may play a role in the degradation of this liver enzyme notably of its many unstable disease-associated mutant forms. The present approach may also have a more general application in the study of liver proteins as possible targets for ubiquitination. (C) 2001 Elsevier Science B.V. All rights reserved.