Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogues of chorismate.
Chorismate-5,6-epoxide, chorismate-5,6-diol, various adamantane derivatives and 2-hydroxy-phenyl acetate are structural analogues of chorismate that act as competitive inhibitors of both the chorismate mutase and prephenate dehydrogenase activities of the bifunctional enzyme, hydroxyphenylpyruvate synthase. The interactions of these chorismate analogues with both activities of the synthase are investigated further. Chorismate mutase and prephenate dehydrogenase activities were assayed spectrophotometrically at 290 and 340 nm, respectively. Data were fit by non-linear regression to appropriate equations describing the time-dependent formation of product or decay of enzymic activity. In the presence of these chorismate analogues, both the mutase and dehydrogenase activities undergo a time-dependent partial inactivation. Progress curves for synthesis of product by the mutase or dehydrogenase in the presence of chorismate-5,6-epoxide, chorismate-5,6-diol or adamantane-1,3-diacetate resemble time-courses characteristic of slow-binding inhibition. However, if the bifunctional enzyme was preincubated with a chorismate analogue prior to addition of substrate, only a minor proportion of enzymic activity was recovered, excluding the possibility of reversible, slow-binding inhibition. When hydroxyphenylpyruvate synthase binds certain chorismate analogues to form an EI complex, there is a slow conformational transition to an ET complex, which may be susceptible to oxidation leading to partial inactivation. Some protection against this inactivation is provided by high concentrations of dithiothreitol (20 mM), suggesting that the inactivation may be due to chemical oxidation.
Christopherson
R I
RI
Department of Biochemistry, University of Sydney, NSW, Australia.
D000218:Adamantane / Q000031:analogs & derivatives / Q000494:pharmacology*; D002826:Chorismate Mutase / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D011303:Prephenate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011303:Prephenate Dehydrogenase; D002826:Chorismate Mutase; D000218:Adamantane