Substrate-assisted catalysis in cytochrome P450eryF

A highly conserved threonine in the active site of cytochromes P450 has been proposed to participate in O-2 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.