STUDIES WITH FLAVIN ANALOGS PROVIDE EVIDENCE THAT A PROTONATED REDUCED FMN IS THE SUBSTRATE-INDUCED TRANSIENT INTERMEDIATE IN THE REACTION OF ESCHERICHIA-COLI CHORISMATE SYNTHASE

Chorismate synthase catalyzes the 1,4-elimination of phosphate and the C-(6-pro-R) hydrogen from 5-enolpyruvylshikimate S-phosphate (EPSP) t o generate chorismate, Although this reaction does not involve an over all change in redox state, the enzyme requires reduced FMN. To investi gate the role of the flavin in catalysis we have employed chemically m odified flavins: 1- and 5-deaza-, 2- and 4-thio-, 6-hydroxy-, 8-nor-6- methyl-, 8-methyl-sulfonyl-, 8-chloro-, 8-fluoro-, 8-nor-methyl-, 8-S- methyl-, 8-methoxy, 8-mercapto- and 8-amino-FMN. Photoreduction of 4-t hio-FMN in the presence of chorismate synthase at pH 7.5 produced a re duced flavin species with an absorbance maximum at lambda = 410 nm ind icative of monoanionic, reduced 4-thio-FMN. Binding of 8-mercapto- and 6-hydroxy-FMN to chorismate synthase in the presence of EPSP or (6R)- 6-fluoro-EPSP resulted in an increase of the flavin analogs' pK(a) val ues by 4 and 1 pH units, respectively, On the basis of these findings it is concluded that chorismate synthase preferentially binds neutral flavin species, including the protonated reduced form, rather than ani onic flavin species in the presence of EPSP or the 6-fluoro-substrate analog, Further support for this conclusion was obtained using 5-deaza - and 4-thio-FMN, Addition of EPSP to enzyme bound, reduced 5-deaza-FM N produced spectral changes consistent with protonation of the flavin, Photoreduction of 4-thio-FMN in the presence of enzyme and the (6R)-6 -fluoro-EPSP generated a reduced flavin species with absorbance proper ties of a neutral, reduced 4-thio-flavin. These results and their impl ications for the nature and kinetic properties of an observed flavin i ntermediate are discussed in the context of a possible role of reduced flavin as an electron donor to bound EPSP.