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' (DeltaN102/DeltaC24-hPAH) as visualized by two-dimensional electrophoresis. The ubiquitination of wt-PAH 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.