Substrate-assisted catalysis in cytochrome P450eryF.
A highly conserved threonine in the active site of cytochromes P450 has been proposed to participate in O2 binding and cleavage. Cytochrome P450eryF is unusual in having alanine in place of this threonine and an ordered active site water molecule (Wat 519) which is hydrogen bonded to the substrate 5-hydroxyl group and is in position to operate as an acid catalyst required for cleaving dioxygen. To asses the role of this alanine residue and Wat 519 in catalysis, two mutant forms of P450eryF (Ala --> Ser,Ala --> Thr) and a substrate analogue lacking a 5-hydroxyl group were examined using kinetic, spectral and crystallographic techniques. In each case decreased catalytic activity was correlated with a loss or repositioning of Wat 519. These findings suggest that P450eryF utilizes the substrate to assist in the acid-catalysed dioxygen bond cleavage reaction.
Cupp-Vickery
J R
JR
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.
Han
O
O
Hutchinson
C R
CR
Poulos
T L
TL
D001426:Bacterial Proteins; D002384:Catalysis; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006860:Hydrogen Bonding; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008961:Models, Structural; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D010100:Oxygen