Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.
It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.
Kowlessur
D
D
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Yang
X J
XJ
Kaufman
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine
D017931:DNA Primers; D008244:Lysophosphatidylcholines; D011994:Recombinant Proteins; D012694:Serine; D010651:Phenylalanine Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases