Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

Phenylalanine hydroxylase (PAH) is a non-heme iron dioxygenase catalyzing the conversion of phenylalanine to tyrosine and is present in both prokaryotic and eukaryotic organisms. A relatively simple PAH is expressed by Chromobacterium violaceum, a gram-negative bacterium found in tropical and subtropical regions. The effects of temperature, pH and metals on the stability and catalytic activity of Chromobacterium violaceum PAH were determined by steady-state kinetics, circular dichroism (CD) and differential scanning calorimetry (DSC). The k(cat) and K-M for phenylalanine were determined between 7 and 40 degreesC. The K-M remained constant between 20 and 40 degreesC but rapidly increased below 20 degreesC. The half-life of the enzyme at 47 degreesC is 66 +/- 4 min in the presence of Fe(II) and 8 +/- 1 min in the presence of EDTA. The melting temperature of the protein determined by CD and DSC is 53 +/- 2 degreesC in the presence of EDTA and 63 +/- 2 degreesC in the presence of Fe(II). Co(II) stabilizes the enzyme (T-m = 63 +/- 2 degreesC) and inhibits the catalytic activity by displacing iron from the active site. The optimum pH for catalytic activity and stability is 7.4. In conclusion, PAH is adapted for optimal phenylalanine binding at temperatures above 20 degreesC and Fe(II) enhances the resistance of the enzyme to thermal denaturation. (C) 2004 Elsevier Inc. All rights reserved.