MECHANISTIC STUDIES ON CDP-6-DEOXY-DELTA(3,4)-GLUCOSEEN REDUCTASE - THE ROLE OF CYSTEINE RESIDUES IN CATALYSIS AS PROBED BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

CDP-6-deoxy-Delta(3,4)-glucoseen reductase (E(3)), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-a scarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yers inia pseudotuberculosis, has been expressed at high level in Escherich ia coli (670 times over the wild-type strain), This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactiv ated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimid e. In both cases the inactivation followed a pseudo first order kineti cs, The second order rate constant for the reaction of DTNB with E(3) was 0.25 mM(-1) min(-1) at 20 degrees C, pH 8.0. Detailed characteriza tion of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivat ion was reversible by treating the inactivated enzyme with 1 mM D,L-di thiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the m ethod developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E(3) 's activity. Tryptic digestion of E(3) labeled with N-ethyl[2,3-C-14]m aleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were s eparately isolated as a pair of interconvertible diastereoisomers. Seq uence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comp arable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD(P) binding domain of enzymes belonging to the same class, this residue may be involved in stabilizing the cha rge-transfer complex between E(3) and NADH, thus facilitating hydride transfer from the nicotinamide nucleotide to flavin. A chemically modi fied Cys-75 which is immediately adjacent to the [2Fe-2S] center in E( 3) may prevent the proper juxtaposition of the redox centers and thus impede electron transfer leading to enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprisi ng the active site of E(3) for electron transfer between NADH, FAD, an d the [2Fe-2S] center.