Structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis.
Phenylalanine hydroxylase (PAH) purified from rat liver is an oligomeric protein (predominantly tetramers) composed of 52-kDa subunits that are identical in primary structure. We have used radiation target analysis to probe the subunit organization of the enzyme. When 6-methyltetrahydropterin was used as the cofactor, the loss of hydroxylase activity as a function of radiation dose was defined by a single exponential decay, yielding a target size of about 120 kDa. However, when the enzyme was assayed with the natural cofactor tetrahydrobiopterin (BH4), the inactivation curves were much more complex. In these cases, the activity first increased, then decreased, as a function of radiation dose. The inactivation profile at higher radiation doses implied a target size of approximately 100 kDa. Kinetic analysis of the enzyme was significantly activated relative to the nonirradiated sample. In addition, the irradiated enzyme was desensitized to substrate-level activation by phenylalanine. The initial increase in activity at low radiation doses is due to the destruction of a large inhibitor. Analysis of the irradiated samples by high-performance size-exclusion chromatography indicated that the hydroxylase tetramer was lost with a target size of 110 kDa. Our data indicate that the tetrameric form of purified PAH consists of two enzymatically active dimers and that BH4 interacts with a tetramer to inhibit or deactivate the enzymatic activity.
Davis
M D
MD
National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
Parniak
M A
MA
Kaufman
S
S
Kempner
E
E
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D008970:Molecular Weight; D010651:Phenylalanine Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation; D011622:Pterins / Q000378:metabolism; D011829:Radiation Dosage; D051381:Rats
D011622:Pterins; D001708:Biopterins; C017523:6-methyltetrahydropterin; D010651:Phenylalanine Hydroxylase; C003402:sapropterin