Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.
Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.
Citron
B A
BA
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892.
Davis
M D
MD
Kaufman
S
S
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D005822:Genetic Vectors; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000096:biosynthesis / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D008244:Lysophosphatidylcholines; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D004247:DNA; D010651:Phenylalanine Hydroxylase