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FINDINGS BY CHEMIST JOHN CARADONNA MAY LEAD TO TARGETED THERAPEUTICS FOR PKU
John Caradonna, associate professor of chemistry at Boston University, together with Edward Solomon and other researchers at Stanford University, has shown that the incorrect binding of a chemical helper, known as a pterin, is responsible for perturbing the enzyme’s action. This chemical cofactor works with the enzyme to maintain correct levels of the amino acid phenylalanine in the body. The enzyme involved in this conversion process is called phenylalanine hydroxylase or PAH. PKU is an inherited disease. It occurs when a gene that carries instructions for PAH is changed, producing a mutated enzyme that works partially or not at all. Without a well-working enzyme, phenylalanine concentrations rise to levels that harm the brain, leading to developmental delays in infants and, by six months, signs of mental retardation. Since the 1960s, when a blood test for PKU was developed and adopted for use in most states, infants with the disease have been diagnosed and treated from birth. Treatment consists of a strictly managed, life-long diet that eliminates meat, milk, nuts, and other foods that contain phenylalanine. Caradonna, a bioinorganic chemist, deciphers the molecular mechanisms of enzymes like PAH, a class of molecules having an iron atom that serves as the active site for reactions in which molecular oxygen (O2) is split. For their study, Caradonna and his collaborators compared the structural and activity characteristics of normal PAH with that of two mutant forms of the enzyme. As with many of the mutant enzymes associated with PKU, the enzymes used by the researchers convert phenylalanine to tyrosine but at a lower-than-normal rate (less than 20 percent). The researchers set out to find the molecular basis of this inefficiency. They did so by investigating the characteristics of substrate, enzyme, and cofactor at the starting point for the reaction: the enzyme’s active site. Enzymes act on substrate compounds to facilitate their metabolism in the body. Their activity often involves cofactors. These cofactors usually begin their work by binding with the enzyme in a region known as the active site. This pocket-like area also hosts the substrate molecule, making it a type of pit stop in which chemical changes involving cofactor, substrate, and enzyme can take place quickly, efficiently, and in tightly structured synchrony. Detailed spectroscopic studies examining the influence of both substrate and cofactor binding in the active site of normal PAH have shown that their combined presence changes the geometry of the active site iron from a distorted octahedron to a square pyramidal structure through the loss of a water molecule bound to the iron center. Research indicates the pterin cofactor then reacts with molecular oxygen to form a peroxy–pterin compound. This compound, guided by the conformation of the enzyme’s active site, aligns to form a “bridge” compound with the enzyme’s iron atom through the site previously occupied by the water molecule. The iron–OO–pterin intermediate then undergoes heterolytic cleavage of the O–O bond to generate an iron-oxo species, which transfers a resulting oxygen atom to the phenyalanine substrate to produce tyrosine. The cleaving also produces a modified pterin compound that disassociates from the active site and is reduced, ready to react again with oxygen to produce a new peroxy–pterin compound and restart the conversion process. When the researchers performed similar studies of the active sites of the PKU-inducing mutants, they found some important differences in the pathway mechanism. Although the mutants form peroxy–pterin compounds at a like or faster rate than normal PAH and have similar active sites, the peroxy–pterin species often fail to react with the enzyme’s iron atom. In addition, this thwarted reaction produces a molecule of hydrogen peroxide, which can readily decompose to generate free radical compounds that can either inactivate PAH by irreversibly modifying its active site or, if allowed to escape from the active site, disrupt chemical reactions in the body. Because the active-site iron environment and the positioning of phenylalanine in the active site appear to be unaltered in the mutants, the researchers concluded the differences were the result of improper positioning of the pterin within the active sites of the mutants. By isolating this molecular misalignment, the researchers open the possibility of producing pterin derivatives that could correct this alignment, thereby improving the rate and efficiency of phenylalanine conversion. Their research appears in the May 14 issue of the Journal of the American Chemical Society and can be accessed online at http://pubs.acs.org/cgi-bin/article.cgi/jacsat/2003/125/i19/html/ja029106f.html
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