Homoserine dehydrogenase-I (Escherichia coli): action of monovalent ions on catalysis and substrate association-dissociation.
Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na+ for the normal activating monovalent ion, K+, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [14C]Hse<-->ASA and [3H]NADP+<-->NADPH exchanges were compared in the presence of K+ vs Na+, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K+ and Na+ enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na+ for K+ shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na+ than K+, but that the Na(+)-enzyme is 10-fold less active and exhibits higher substrate Km values, especially for L-Hse.
Wedler
F C
FC
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Ley
B W
BW
D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001225:Aspartokinase Homoserine Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D002414:Cations, Monovalent / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006714:Homoserine / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009249:NADP / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology
D002414:Cations, Monovalent; D001224:Aspartic Acid; C006644:aspartic semialdehyde; D009249:NADP; D006714:Homoserine; D012964:Sodium; D001225:Aspartokinase Homoserine Dehydrogenase; D011188:Potassium