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 kcat and KM for phenylalanine were determined between 7 and 40 degrees C. The KM remained constant between 20 and 40 degrees C but rapidly increased below 20 degrees C. The half-life of the enzyme at 47 degrees C 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 degrees C in the presence of EDTA and 63+/-2 degrees C in the presence of Fe(II). Co(II) stabilizes the enzyme (Tm=63+/-2 degrees C) 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 degrees C and Fe(II) enhances the resistance of the enzyme to thermal denaturation.
Zoidakis
Jérôme
J
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Loaiza
Aristobulo
A
Vu
Kim
K
Abu-Omar
Mahdi M
MM
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002861:Chromobacterium / Q000201:enzymology*; D002942:Circular Dichroism; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013696:Temperature
D005296:Ferrous Compounds; D008670:Metals; D004492:Edetic Acid; D010651:Phenylalanine Hydroxylase