Enzyme Mechanisms

For 40 years the biosynthesis of prostaglandins has been thought to proceed through an all radical mechanism.  Although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, we show that the available evidence demonstrates it effects cyclizations through a carbocation.

Dean & Dean. 1999. Protein Science 8:1087-1098.

E. coli isocitrate dehydrogenase (IDH) is regulated by a bifunctional protein, IDH kinase/phosphatase.  We show that effectors controlling these activities belong to two distinct classes.  NADPH and isocitrate are representative members of the first class.  NADPH inhibits both IDH kinase and IDH phosphatase.  Isocitrate inhibits only IDH kinase and “activates” IDH phosphatase.  Mutations in IDH produce parallel effects on ligand binding to IDH and on kinase and phosphatase kinetics.  Hence, NADPH and isocitrate regulate IDH kinase/phosphatase through the IDH active site.  AMP, 3-phosphoglycerate, and pyruvate represent the second class of effectors.  These bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme.

Miller, Chen, Karshina, Dean & LaPorte. 2000. J Biol Chem 275:833-839.