Probing the Role of Crystallographically Defined/Predicted Hinge-bending Regions in the Substrate-induced Global Conformational Transition and Catalytic Activation of Human Phenylalanine Hydroxylase by Single-site Mutagenesis

Abstract

Phenylalanine hydroxylase (PAH) is generally considered to undergo a large and reversible conformational transition upon L-Phe binding, which is closely linked to the substrate-induced catalytic activation of this hysteretic enzyme. Recently, several crystallographically solvent-exposed hinge-bending regions including residues 31-34, 111-117, 218 -226, and 425-429 have been defined/ predicted to be involved in the intra-protomer propagation of the substrate-triggered molecular motions generated at the active site. On this basis, single-site mutagenesis of key residues in these regions of the human PAH tetramer was performed in the present study, and their functional impact was measured by steadystate kinetics and the global conformational transition as assessed by surface plasmon resonance and intrinsic tryptophan fluorescence spectroscopy. A strong correlation (r 2 ‫؍‬ 0.93-0.96) was observed between the L-Pheinduced global conformational transition and V max values for wild-type human PAH and the mutant forms K113P, N223D, N426D, and N32D, in contrast to the substitution T427P, which resulted in a tetrameric form with no kinetic cooperativity. Furthermore, the flexible intra-domain linker region (residues 31-34) seems to be involved in a more local conformational change, and the biochemical/biophysical properties of the G33A/G33V mutant forms support a key function of this residue in the positioning of the autoregulatory sequence (residues 1-30) and thus in the regulation of the solvent and substrate access to the active site. The mutant forms revealed a variably reduced global conformational stability compared with wild-type human PAH, as measured by thermal denaturation and limited proteolysis.

Activation of Phenylalanine Hydroxylase Induces Positive Cooperativity toward the Natural Cofactor

“…These variations may be due to the different methods used, yet additional aspects such as different time frames for measurement of steady state kinetics and the improvement of protein purification methods in our laboratory within the last years play an important role and may further explain these findings. Notably, large differences in enzyme activities, ranging up to 7-fold, can also be found in the literature (16,37,52,53). However, kinetic parameters describing apparent affinity and enzyme allostery (S 0.5 , K m /C 0.5 , Hill coefficient (h), and activation-fold) were similar to results obtained using discontinuous assays and to results previously described (17,37), confirming the accuracy of our newly developed continuous assay.…”

“…Using two different spectroscopic techniques we analyzed local and global effects on protein structure by mutation/truncation or by L-Phe, respectively, and correlated this with enzyme kinetic parameters. Preincubation of wild-type PAH with L-Phe leads to a series of structural and functional changes resulting in enzyme activation (16,37,(41)(42)(43)(44)(45). On the structural level these include a red-shifted and enhanced tryptophan emission and a right-shifted thermal denaturation profile.…”

“…Structural analyses of BH 4 binding revealed that the cofactor interacts with the N-terminal autoregulatory sequence and the pterin binding loop, leading to stabilizing hydrogen bonds and to formation of a binary enzyme-BH 4 complex (14). The largest conformational changes were observed upon binding of L-Phe, where local changes at the active site are propagated globally through hinge-bending motions in the catalytic domain, also altering the position and orientation of bound BH 4 (13,15) and of the regulatory domain (16).…”

Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations

“…The cofactor and cosubstrate BH 4 , on the contrary, acts as a negative regulator, blocking the L-Phe-binding site (43). Recent structural and mutational studies of hinge-bending regions (41,42,44) have revealed further evidence that global activating conformational changes caused by L-Phe binding have the epicenter at the active site and are transmitted throughout the enzyme through hinge-bending motions. A reduction or loss of the cooperative substratedependent activation has been seen for most of the kinetically characterized PKU mutations to date (20,45,46), severe as well as mild forms, suggesting that the enzyme is quite susceptible to mutations that destroy the cooperative activation mechanism probably by hindering the transmission of the conformational change.…”

Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

“…Moreover, the observed effect seems to be similar to the one induced by L-Phe binding to the protein. This effect has been reported to cause large conformational changes in the catalytic domain [26,[37][38][39], which in turn are believed to trigger large-scale structural changes throughout the entire protomer, including a displacement of the autoregulatory sequence, which partly covers the active site in the resting state, resulting in a catalytically high activity state of hPAH.…”