Electrostatic activation of rat phenylalanine hydroxylase.
The conversion of phenylalanine to tyrosine is accelerated approximately five fold by phosphorylation of the enzyme which catalyzes this step, phenylalanine hydroxylase. To gain a clearer understanding of the mechanism of this activation, we have applied site-directed mutagenesis to specifically modify a clone of the hydroxylase at the phosphorylation site, the serine at position 16. We converted this serine residue to alanine and to glutamic acid. The wild-type and mutant proteins were purified and the activation states of the enzymes were examined with respect to the single phosphorylation site at position 16. Substitution of Ser16 with a negatively charged Glu residue resulted in activation of the enzyme, whereas substitution with an uncharged Ala residue did not. These results indicate that activation of the native enzyme by phosphorylation is due to the introduction of a negative charge, and suggest involvement of electrostatic interactions.
Citron
B A
BA
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892.
Davis
M D
MD
Kaufman
S
S
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004563:Electrochemistry; D004789:Enzyme Activation; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine
D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D004247:DNA; D010651:Phenylalanine Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine