MECHANISTIC STUDIES ON CDP-6-DEOXY-L-THREO-D-GLYCERO-4-HEXULOSE 3-DEHYDRASE - IDENTIFICATION OF HIS-220 AS THE ACTIVE-SITE BASE BY CHEMICALMODIFICATION AND SITE-DIRECTED MUTAGENESIS

CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E(1)) purified f rom Yersinia pseudotu- berculosis is a pyridoxamine 5'-phosphate (PMP) dependent iron-sulfur-containing enzyme which catalyzes the C-O bond cleavage at C-3 of its substrate leading to the formation of 3,6-dideo xyhexose. This enzyme is rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The inactivation of E(1) by DEP, whi ch is reversible upon treatment of hydroxylamine, appears to be attrib utable solely to the modification of histidine residues. The fact that coincubation of E(1) with its substrate gave almost total protection against DEP inactivation and that only one less histidine residue was modified in the presence of substrate strongly suggested that inactiva tion is due to the modification of only one reactive histidine residue which resides in or near the active site of E(1) and is critical for E(1)'s activity. Sequence alignment between the translated ascC (Ei) g ene and several representative pyridoxal 5'-phosphate (PLP)/PMP depend ent enzymes revealed that three of the four invariant residues, glycin e, aspartate, and arginine found in all other aminotransferases, are c onserved in the E(1) sequence (G169, D191, and R403). However, the hig hly conserved lysine is replaced by a histidine residue (H220) in E(1) . In order to test whether H220 plays an essential role in E(1) cataly sis, H220N mutant was constructed and the encoding protein was found t o exhibit nearly identical physical characteristics as the wild-type E (1). Interestingly, the mutant protein had lost most of its catalytic activity, and one less histidine residue was modified upon treatment o f H220N-mutated protein with DEP. Such a single-point mutation also im paired El's capability of catalyzing the solvent hydrogen exchange at C-4' position of the PMP coenzyme. Our findings strongly suggested tha t H220 is most likely the active-site base which abstracts the C-4' pr oton from the PMP-substrate adduct and initiates the catalysis. Furthe rmore, E(1)'s preservation of other invariant residues found in many P LP/PMP dependent enzymes allowed a speculation of their roles in E(1) catalysis. Since sequence alignment between E(1) and its homologs beli eved to participate as PLP/PMP dependent aminotransferases in the bios ynthesis of deoxy amino sugars showed that substitution of the active- site lysine with a histidine residue seems to be characteristic for st rictly PMP dependent enzymes, it is thus conceivable that nature's sim ple substitution of an active-site lysine with a histidine residue may have transformed a normal coenzyme Bg dependent aminotransferase into a unique PMP dependent catalyst that is no longer an aminotransferase but instead a dehydrase.