The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties.
Pure phenylalanine hydroxylase from rat liver can be activated by limited proteolysis with alpha-chymotrypsin. As with most other types of activation of this enzyme, including activation by exposure to lysolecithin, the increase in activity is expressed in the presence of the naturally occurring pterin cofactor, tetrahydrobiopterin, but not in the presence of synthetic pterin cofactors such as 6-methyltetrahydropterin. With the chymotrypsin-activated enzyme, we have demonstrated directly, using circular dichroism measurements, that the activated enzyme differs in conformation from the native enzyme. In addition to chymotrypsin, trypsin and a mixture of rat liver lysosomal proteases can also activate phenylalanine hydroxylase. The latter finding raises the possibility that activation of the enzyme by limited proteolysis may be a physiologically important process. In experiments carried out with phenylalanine in which all five hydrogens on the aromatic ring have been replaced with deuterium, and in the presence of tetrahydrobiopterin, we have been unable to detect a kinetic isotope effect with either the native hydroxylase or with the hydroxylase activated by limited proteolysis, or by exposure to lysolecithin. By contrast, with both native and activated enzymes, a small isotope effect was detected when 6-methyltetrahydropterin was used as the pterin cofactor.
D000818:Animals; D002918:Chymotrypsin / Q000494:pharmacology*; D002942:Circular Dichroism; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013816:Thermodynamics; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000031:analogs & derivatives*
D008244:Lysophosphatidylcholines; D014810:Vitamin E; C012552:alpha-tocopherol phosphate; D010651:Phenylalanine Hydroxylase; D002918:Chymotrypsin; D024502:alpha-Tocopherol