Substrate analogs as mechanistic probes for the bifunctional chorismate synthase from Neurospora crassa.
Analogs of EPSP (4-8) have been prepared, and their activity as substrates for the chorismate synthase from Neurospora crassa has been characterized kinetically. The enzyme appears to show strict discrimination against substitution at the Z-position of the enol ether side chain as well as against substitution at the S-position of the reduced analogs. Both the glycolyl and (R)-lactyl analogs 4 and (R)-5 are good substrates, with (R)-5 having a higher V value than the natural substrate. Three substrates, including EPSP, have been found to show significant substrate inhibition with this enzyme, which at present can be explained by a noncompetitive model involving formation of a catalytically incompetent, ternary ES2 complex. A significant secondary kinetic isotope effect on V of 1.10 +/- 0.02 has been observed at C-3 with EPSP, indicating that C-O bond cleavage is kinetically significant at saturating substrate concentration; this effect is severely depressed at limiting substrate, with D(V/K) = 0.97 +/- 0.02. A similar effect is found for the primary deuterium isotope effect at C-6R, as observed previously [Balasubramanian, S., Davies, G. M., Coggins, J. R., & Abell, C. (1991) J. Am. Chem. Soc. 113, 8945-8946]. The primary isotope effects at C-6R with reduced analogs (R)-5 and (S)-6 are significantly larger than those with EPSP. The larger values of V and DV for (R)-5, when compared to EPSP, are evidence that release of chorismate is partially rate-limiting under saturating conditions. Incubation of the enzyme with reduced 5-deazaFMN does not result in any observable formation of chorismate, consistent with previous results indicating that reduced flavin is chemically involved in the synthesis of chorismate from EPSP [Ramjee, M. N., Balasubramanian, S., Abell, C., Coggins, J. R., Davies, G. M., Hawkes, T. R., Lowe, D. J., & Thorneley, R. N. F. (1992) J. Am. Chem. Soc. 114, 3151-3153].
Lauhon
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Department of Chemistry, University of California, Berkeley 94720-1460.
Bartlett
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D003903:Deuterium; D005415:Flavins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015394:Molecular Structure; D009492:Neurospora crassa / Q000201:enzymology*; D009711:Nucleotides / Q000494:pharmacology; D019761:Phosphorus-Oxygen Lyases; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005415:Flavins; D009711:Nucleotides; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D003903:Deuterium; D008190:Lyases; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases