REEVALUATING GLYPHOSATE AS A TRANSITION-STATE INHIBITOR OF EPSP SYNTHASE - IDENTIFICATION OF AN EPSP SYNTHASE-CENTER-DOT-EPSP-CENTER-DOT-GLYPHOSATE TERNARY COMPLEX

Numerous studies have confirmed that glyphosate forms a light ternary complex with EPSP synthase and shikimate 3-phosphate. It has been prop osed [Anton. D., Hedstrom, L., Fish, S., and Abeles, R. (1983) Biochem istry 22, 5903-5908; Steinrucken. H. C., &: Amrhein, N. (1984) Eur. J. Biochem. 143, 351-357] that in this complex glyphosate functions as a transition-state analog of the putative phosphoenolpyruvoyl oxonium i on. For this to be true. glyphosate must occupy the space in the enzym e active site that is normally associated with PEP and. through turnov er, the carboxyvinyl group of the product EPSP. According to this mode l, one would predict that, in the reverse EPSP synthase reaction with EPSP and phosphate as substrates, there should bt little if any intera ction of glyphosate with enzyme or enzyme substrate complexes. In cont rast to this expectation, rapid gel filtration experiments provided di rect evidence that glyphosate could be trapped on the enzyme in the pr esence of EPSP to form a ternary complex of EPSPS . EPSP . glyphosate. The experimentally determined stoichiometry for this complex, 0.62 eq uiv of glyphosate/mole of EPSPS, is similar to that found for the EPSP S . S3P . glyphosate ternary complex (0.66). This direct binding resul t was corroborated and quantitated by fluorescence titration experimen ts which demonstrated that glyphosate forms a reasonably tight (K-d = 56 +/- 1 mu M) ternary complex with enzyme and EPSP. This finding was further verified. and its impact on substrate turnover analyzed, by st eady-state kinetics. Glyphosate was found to be an uncompetitive inhib itor versus EPSP with K-ii(app) = 54 +/- 2 mu M. Taking these results together, it is apparent that the carboxyvinyl group in EPSP does not prevent glyphosate binding, and in fact it strongly facilitates the bi nding of this inhibitor to the enzyme. It has been previously demonstr ated that glyphosate has little (K-d = 12 mM) interaction with free en zyme [Ream, J, E., Yuen, H. K., Frazier, R. B., and Sikorski, J. A. (1 992) Biochemistry 31, 5528-5534]. Interestingly, glyphosate was a mixe d inhibitor (K-ii(app) = 18.2 +/- 0.4 mu M, K-ii(app) = 23.9 It 0.3 mu M) versus phosphate, This surprising result suggests that the formati on of an EPSPS . EPSP . glyphosate ternary complex does not preclude p hosphate binding and that a quaternary complex of enzyme containing bo th substrates plus inhibitor can occur. The proposed model for glyphos ate functioning as a transition state analog inhibitor, which has defi ned the: paradigm for glyphosate's molecular mode of action for more t han a decade, is very difficult to reconcile with these results. Glyph osate does not meet the key criteria required of a transition-state an alog or, alternatively. all intermediate mimic inhibitor. Glyphosate, therefore, can no longer be classified as such for this system.