Engineering the active site of ascorbate peroxidase

The oxidation of a number of thioethers, namely methyl phenyl sulphide (1), ethyl phenyl sulphide (2), isopropyl phenyl sulphide (3), n-propyl phenyl sulphide (4), p-chlorophenyl methyl sulphide (5), p-nitrophenyl methyl sulp hide (6) and methyl naphthalene sulphide (7), by recombinant pea cytosolic ascorbate peroxidase (rAPX) and a site-directed variant of rAPX in which th e distal tryptophan 41 residue has been replaced with an alanine (W41A) has been examined. The electronic spectrum (pH 7.0, mu = 0.10 M, 25.0 degreesC ) for the ferric derivative of W41A (lambda (max)/nm = 411, 534, 560, 632) is indicative of an increased quantity of 6-coordinate, high-spin and/or 6- coordinate, low-spin haem compared to rAPX. Steady state oxidation of sulph ides 1-4 and 7, gave values for k(cat) that are approximately 10-fold and 1 00-fold, respectively, higher for W41A than for rAPX. For rAPX, essentially racemic mixtures of R- and S-sulphoxides were obtained for all sulphides. With the exception of sulphide 7, the W41A variant shows substantial enhanc ements in enantioselectivity. with R : S ratios varying between R : S = 63 : 37 (sulphides 1 and 4) and R : S = 85 : 15 (sulphide 6). Incubation of su lphide 2 with rAPX or W41A and [O-18] H2O2 shows 95% (rAPX) and 96% (W41A) transfer of labelled oxygen to the substrate. Structure-based modelling tec hniques have provided a fully quantitative rationalization of all the exper imentally determined R : S ratios and have indicated that reorientation of the sidechain of Arg38, such that access to the haem is much less restricte d, is influential in controlling the stereoselectivity for both rAPX and W4 1A.