期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 12, 页码 4359-4367出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.006477
关键词
allosteric regulation; small-angle X-ray scattering (SAXS); enzyme kinetics; analytical ultracentrifugation; hydroxylase; protein conformation; enzyme stability; phenylalanine hydroxylase; phenylketonuria; allostery; protein structure
资金
- Office of the Vice President for Research at UT Health San Antonio
- National Science Foundation (NSF)
- National Institutes of Health (NIH)/NIGMS via NSF Grant [DMR-1332208]
- NIH/NIGMS [GM-103485]
The naturally occurring R68S substitution of phenylalanine hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of phenylalanine, in contrast to the WT enzyme. Analytical ultracentrifugation establishes that the isolated regulatory domain of R68S PheH is predominantly monomeric in the absence of phenylalanine and dimerizes in its presence, similar to the regulatory domain of the WT enzyme. Fluorescence and small-angle X-ray scattering analyses establish that the overall conformation of the resting form of R68S PheH is different from that of the WT enzyme. The data are consistent with the substitution disrupting the interface between the catalytic and regulatory domains of the enzyme, shifting the equilibrium between the resting and activated forms approximate to 200-fold, so that the resting form of R68S PheH is approximate to 70% in the activated conformation. However, R68S PheH loses activity 2 orders of magnitude more rapidly than the WT enzyme at 37 degrees C and is significantly more sensitive to proteolysis. We propose that, even though this substitution converts the enzyme to a constitutively active enzyme, it results in PKU because of the decrease in protein stability.
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