STRUCTURE AND FUNCTION OF MUTANT ARG44LYS OF 4-HYDROXYBENZOATE HYDROXYLASE - IMPLICATIONS FOR NADPH BINDING

Arg44, located at the si-face side of the flavin ring in 4-hydroxybenz oate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydro xybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hy droxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit be tween Ile43 and Lys44 is flipped by about 180 degrees in 50% of the mo lecules. The phi,psi angles in both the native and flipped conformatio n are outside the allowed regions and indicate a strained conformation . [R44K]4-Hydroxygenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions be tween the side chain of Arg44 and the diphosphoribose moiety of the FA D. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type . [R44K]4-Hydroxygenzoate hydroxylase fully couples flavin reduction t o substrate hydroxylation. Stopped-flow kinetics showed that the effec tor role of 4-hydroxybenzoate is largely conserved in the mutant. Repl acement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NA DP(+). It is inferred from these data that Arg44 is indispensable for optimal catalysis.