Use of site-directed mutagenesis to identify residues specific for each reaction catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli.

Site-directed mutagenesis was performed on the bifunctional enzyme chorismate mutase-prephenate dehydrogenase in order to identify groups important for each of the two reactions. We selected two residues for mutagenesis, Lys37 and His131, identified previously by differential peptide mapping to be essential for activity [Christendat, D., and Turnbull, J. (1996) Biochemistry 35, 4468-4479]. Kinetic studies reveal that K37Q exhibits no mutase activity while retaining wild-type dehydrogenase activity, verifying that Lys37 plays a key role in the mutase. By contrast His131 is not critical for the dehydrogenase; H131A is a reasonably efficient catalyst exhibiting 10% dehydrogenase and 30% mutase activity compared to the wild-type enzyme. Chemical modification of H131A by diethyl pyrocarbonate further inactivated the dehydrogenase, suggesting that a different histidine is now accessible to modification. To identify this group, the protein's remaining eight histidines were changed to alanine or asparagine. A single substitution, H197N, decreased the dehydrogenase activity by 5 orders of magnitude while full mutase activity was retained. In H197N, the Michaelis constants for prephenate and NAD+ and the mutant's elution profile from Sepharose-AMP were similar to those of wild-type enzyme, indicating that catalysis rather than substrate binding is altered. Log V for the dehydrogenase reaction catalyzed by H197N is pH-independent and is in contrast to wild-type enzyme, which shows a decrease in activity at low pH and pK of about 6.5. We conclude that His197 is an essential catalytic residue in the dehydrogenase reaction.