Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor
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Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor. / Gersting, Søren W; Staudigl, Michael; Truger, Marietta S; Messing, Dunja D; Danecka, Marta K; Sommerhoff, Christian P; Kemter, Kristina F; Muntau, Ania C.
In: J BIOL CHEM, Vol. 285, No. 40, 01.10.2010, p. 30686-97.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Activation of phenylalanine hydroxylase induces positive cooperativity toward the natural cofactor
AU - Gersting, Søren W
AU - Staudigl, Michael
AU - Truger, Marietta S
AU - Messing, Dunja D
AU - Danecka, Marta K
AU - Sommerhoff, Christian P
AU - Kemter, Kristina F
AU - Muntau, Ania C
PY - 2010/10/1
Y1 - 2010/10/1
N2 - Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.
AB - Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.
KW - Allosteric Regulation
KW - Biopterin
KW - Coenzymes
KW - Enzyme Activation
KW - Fluorescence
KW - Humans
KW - Kinetics
KW - Mutation, Missense
KW - Phenylalanine
KW - Phenylalanine Hydroxylase
KW - Phenylketonurias
U2 - 10.1074/jbc.M110.124016
DO - 10.1074/jbc.M110.124016
M3 - SCORING: Journal article
C2 - 20667834
VL - 285
SP - 30686
EP - 30697
JO - J BIOL CHEM
JF - J BIOL CHEM
SN - 0021-9258
IS - 40
ER -