CHORISMATE-UTILIZING ENZYMES ISOCHORISMATE SYNTHASE, ANTHRANILATE SYNTHASE, AND P-AMINOBENZOATE SYNTHASE - MECHANISTIC INSIGHT THROUGH INHIBITOR DESIGN

Three enzymes of the shikimic acid pathway, isochorismate synthase (IS ), anthranilate synthase (AS), and p-aminobenzoate synthase (PABS), ex hibit significant sequence homology and may be related mechanistically . Compounds 1, 2, and 3 were designed to mimic, in their all-axial con formations, the putative transition state for these enzymes. The inhib itors were prepared in racemic form starting from Diels-Alder addition of a propiolate ester to a protected 1-oxy- or 1-amino-1,3-butadiene in 14%, 4%, and 9%, overall yields, respectively. All three compounds are competitive inhibitors of the three enzymes, binding IS and AS str ongly and PABS weakly. For both IS and AS, the affinity of the 6-amino -4-hydroxy isomer 2 is ca. 10-fold that of the 4-amino-6-hydroxy isome r 3, a difference that is largely due to their conformational equilibr ia; 2 is 25 +/- 2% axial and 3 is 6 +/- 3% axial, as determined by the temperature dependence of their NI?IR spectra. The similarity between IS and AS was extended by the finding that IS, like AS, catalyzes for mation of 2-amino-2-deoxyisochorismate (ADIC) in the presence of ammon ia. These observations are consistent with direct 1,5-substitution mec hanisms for both IS and AS; the weak inhibition of PABS by these inhib itors suggests that it operates by a significantly different mechanism .