Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites.
The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.
Turnbull
J
J
Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Morrison
J F
JF
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008968:Molecular Conformation; D011303:Prephenate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000494:pharmacology
D001426:Bacterial Proteins; D014443:Tyrosine; D011303:Prephenate Dehydrogenase