Phenylalanine hydroxylase from Chromobacterium violaceum. Purification and characterization

Area of Science:

• Biochemistry
• Enzymology
• Microbial Physiology

Background:

• Phenylalanine hydroxylase (PAH) is a key enzyme in amino acid metabolism.
• Understanding PAH structure and function is vital for metabolic research.
• Chromobacterium violaceum serves as a model organism for studying bacterial enzymes.

Purpose of the Study:

• To purify and characterize phenylalanine hydroxylase from Chromobacterium violaceum.
• To determine the enzyme's physical and kinetic properties.
• To investigate the enzyme's molecular composition.

Main Methods:

• Purification of phenylalanine hydroxylase to homogeneity.
• Crystallization of the enzyme from L-phenylalanine-induced cells.
• Determination of molecular weight, Stokes radius, sedimentation and diffusion coefficients, frictional ratio, and isoelectric point.
• Enzyme kinetics assays to determine Km values for substrates.

Main Results:

• Phenylalanine hydroxylase was purified ~3000-fold with 13% yield.
• The enzyme consists of a single polypeptide chain (MW ~32,000).
• Key physical properties determined: Stokes radius (26.0 A), sedimentation coefficient (2.71 S), diffusion coefficient (8.20 x 10(-7) CM2/S), frictional ratio (1.23), and isoelectric point (pH 4.5).
• No detectable iron was found in the purified enzyme.
• Apparent Km values for L-phenylalanine and the pteridine cofactor were 140 µM and 54 µM, respectively.

Conclusions:

• The purified Chromobacterium violaceum phenylalanine hydroxylase is a homogeneous, single-chain protein.
• Its physical and kinetic properties provide insights into its catalytic mechanism.
• The absence of iron suggests it is not a typical non-heme iron hydroxylase.
• This characterization provides a foundation for further structural and functional studies of PAH.