PH DEPENDENCY OF THE REACTIONS CATALYZED BY CHORISMATE MUTASE PREPHENATE DEHYDROGENASE FROM ESCHERICHIA-COLI

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/K(prephenate), as well as in the V/K(NAD), profile. The V/K(prephenate) profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/K(chorismate) profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the K(i) profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/K(chorismate) profile. By contrast, the K(i) profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor. On the basis of the aforementioned data, hypotheses are proposed for the chemical mechanisms of the prephenate dehydrogenase and chorismate mustase reactions. For the mutase reaction, the data suggest that the groups of chorismate required for the binding of substrate at the active site of the enzyme are the 4-hydroxyl, the ring carboxyl, and the oxygen moiety of the enolpyruvyl side chain. The group binding the latter moiety could also be involved with catalysis.