Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Abstract

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsinlike enzymes and on the increase in catalytic efficiency.

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

“…For example, we still lack information on the conformational space available to the first ∼20 residues. Both NMR and molecular dynamics simulations suggest that the mobile N-terminal peptide can sample two distinct conformations in the absence of Phe, but prefers one of these when phosphorylated (24,25). Mobility in this region is lost upon addition of sufficient Phe to fully activate PAH (25), suggesting that a structure of the fully active enzyme may reveal more information about this region.…”

Dynamic Regulation of Phenylalanine Hydroxylase by Simulated Redox Manipulation

“…Changes in stability were shown by MD simulations upon phosphorylation of Ser-16 [67]. This computational technique was also used to infer factors for specificity and affinity of PAH ligand binding [68], [69].…”

Monitoring of Salt Tolerance Responses of Different Maize Inbred Lines Using Protein Profile and Protein Spectrofluorescence

“…The other important observation is in case of losing the quenching effect the salt stress protein samples of varieties L1 and L2 show higher peak intensity rather than the average intensity of control samples. Several data show increase of protein fluorescence intensity as a result of protein phosphorylation process or protein binding with calcium (Miranda et al, 2004;VanScyocet al, 2002). Under salt stress, plants activate sensor proteins through protein binding with calcium and phosphorylation of many protein kinases (Du et al, 2011).…”