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J. Biol. Chem., Vol. 279, Issue 25, 26571-26580, June 18, 2004

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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^*

Anne Jorunn Stokka, Raquel Negrão Carvalho, João Filipe Barroso, and Torgeir Flatmark

From the Section of Biochemistry and Molecular Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway

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 steady-state kinetics and the global conformational transition as assessed by surface plasmon resonance and intrinsic tryptophan fluorescence spectroscopy. A strong correlation (r² = 0.93-0.96) was observed between the L-Phe-induced 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.

Received for publication, January 27, 2004 , and in revised form, March 31, 2004.

^* This work was supported in part by grants from the Norwegian Research Council (to A. J. S. and T. F.), the Fundação para a Ciência e Tecnologia, Portugal, Grants SFRH/BD/1100/2000 (to R. N. C.) and PRAXIS XXI/BD/21690/99 (to J. F. B.), the University of Bergen (to A. J. S. and T. F.), the Novo Nordisk Foundation, the Blix Family Fund, and Rebergs Legat (to T. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 47-55-586428; Fax: 47-55-586360; E-mail: torgeir.flatmark{at}biomed.uib.no.

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