title: Production and characterization of bifunctional enzymes. Substrate channeling in the aspartate pathway.
abstract: The direct channeling of an intermediate between enzymes that catalyze consecutive reactions in a pathway offers the possibility of an efficient, exclusive, and protected means of metabolite delivery. Aspartokinase-homoserine dehydrogenase I (AK-HDH I) from Escherichia coli is an unusual bifunctional enzyme in that it does not catalyze consecutive reactions. The potential channeling of the intermediate beta-aspartyl phosphate between the aspartokinase of this bifunctional enzyme and aspartate semialdehyde dehydrogenase (ASADH), the enzyme that catalyzes the intervening reaction, has been examined. The introduction of increasing levels of inactivated ASADH has been shown to compete against enzyme-enzyme interactions and direct intermediate channeling, leading to a decrease in the overall reaction flux through these consecutive enzymes. These same results are obtained whether these experiments are conducted with aspartokinase III, a naturally occurring monofunctional isozyme, with an artificially produced monofunctional aspartokinase I, or with a fusion construct of AK I-ASADH. These results provide definitive evidence for the channeling of beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in E. coli and suggest that ASADH may provide a bridge to channel the intermediates between the non-consecutive reactions of AK-HDH I.
authors.affiliation: Department of Chemistry, University of Toledo, Toledo, Ohio 43606, USA.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: James
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism*; D001225:Aspartokinase Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000138:chemical synthesis* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000138:chemical synthesis* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D001224:Aspartic Acid; D001225:Aspartokinase Homoserine Dehydrogenase