Probing cofactor specificity in phenylalanine hydroxylase by molecular dynamics simulations.

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine using dioxygen as an additional substrate. The requirement of PAH for a cofactor is absolute, but several cofactor analogs are able to substitute the natural cofactor in catalysis. However, it is only the natural cofactor 6R-tetrahydrobiopterin (6R-BH(4)) that induces a negative regulatory effect on the enzyme. In order to get further insights on the molecular basis for this specificity, we studied the structure of the cofactor-enzyme complex and the conformational changes induced by cofactor binding by molecular dynamics simulations. Simulations were carried out on the enzyme alone and complexed with 6R-BH(4) and with two cofactor analogs, 6S-BH(4) and 6-methyl-tetrahydropterin (6M-PH(4)). In the resting unbound enzyme Tyr377 in the catalytic domain is hydrogen bonded to both Ser23 and Glu21 of the autoregulatory N-terminal sequence. This hydrogen bonding network is disturbed by the binding of BH(4), which interacts with Ser23. By doing so, 6R-BH(4) facilitates an interaction between Glu21 and the active site iron, further pulling the N-terminal into the active site of PAH and blocking the L-Phe binding site. Thus, in the 6R-BH(4) complexed enzyme, the N-terminal functions as an intrinsic amino acid regulatory sequence (IARS). Neither 6M-PH(4) nor 6S-BH(4) can interact favorably with Ser23, and do not induce an inhibitory effect on PAH. These simulations thus explain the previous findings that the two hydroxyl groups in the side chain of the 6R epimer of BH(4) are essential for the inhibitory regulatory effect on PAH.