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Protein Science (2004), 13:1219-1226. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Frederico Faria Miranda1,2, Matthías Thórólfsson1, Knut Teigen1, Jose M. Sanchez-Ruiz3 and Aurora Martínez1

1 Department of Biomedicine, University of Bergen, 5009-Bergen, Norway
2 Department of Biochemistry, Universidade de Coimbra, 3000 Coimbra, Portugal
3 Departamento de Química Física, Facultad de Ciencias, 18071 Granada, Spain

(RECEIVED December 28, 2003; FINAL REVISION February 4, 2004; ACCEPTED February 9, 2004)



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 trypsin-like enzymes and on the increase in catalytic efficiency.

Keywords: phosphorylation; phenylalanine hydroxylase; molecular dynamics simulations; conformational stability; electrostatic interactions; proteolysis

Abbreviations: BH4, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin • CD, circular dichroism • DSC, differential scanning calorimetry • MD, molecular dynamics • PAH, phenylalanine hydroxylase • PKA, cyclic AMP–dependent protein kinase • wt-PAH, wild-type phenylalanine hydroxylase.

Reprint requests to: Aurora Martínez, Department of Biomedicine, Jonas Lies vei 91, 5009-Bergen, Norway; e-mail: aurora.martinez{at}ibmb.uib.no; fax: 47-55586360.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03595904.


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