Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Shengnan Zhang,Paul F Fitzpatrick

doi:10.1074/jbc.m115.709998

Abstract

Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

Highlights

Mammalian phenylalanine hydroxylase (PheH) is a homotetramer, and each monomer contains an N-terminal regulatory domain, a central catalytic domain, and a C-terminal tetramerization domain
We report that the isolated regulatory domain of PheH (RDPheH) forms a side-by-side ACT domain dimer, with helix ␣1, strand ␤2, and the loops connected to strand ␤2 forming the dimer interface and that phenylalanine binds across the two edges of the dimer interface
The results provide further support for a regulatory mechanism in which phenylalanine binding to the regulatory domain of PheH is linked to regulatory domain dimerization and that this is the conformational change associated with activation

Summary

Experimental Procedures

Materials—2H8-L-Phenylalanine, 15NH4Cl, 13C6-D-glucose, D-glucose-1,2,3,4,5,6,6-d7, and deuterium oxide were from Cambridge Isotope Laboratories, Inc. (Andover, MA). For 15N-labeled or 15N/13C-labeled RDPheH[25–117], the expression was the same as that for the unlabeled proteins except that the cells were grown in M9 minimal media with 15NH4Cl (1 g/liter) in the absence or presence of 13C6-glucose (4 g/liter), respectively [27]. Stopped-flow Spectroscopy—The binding of phenylalanine to different PheH variants was monitored using fluorescence spectroscopy as described previously [25]. The total fluorescence change as a function of the concentration of phenylalanine was fit to Equation 2,. Enzyme Assays—The effects of preincubation with phenylalanine on the activities of different PheH mutants were determined as described previously for wild-type PheH [25]. Steady-state kinetic parameters were determined by fitting the rates as a function of phenylalanine concentration to Equation 3, v e ϭ kcat K0n.5 ϫ ϩPhenPhen (Eq 3). Where n is the Hill coefficient; e is the enzyme concentration, and K0.5 is the concentration of phenylalanine at which the rate is one-half of kcat

Results

Kinetic parameters for PheH variants

Discussion