Rate limitations in the lumazine synthase mechanism

Lumazine synthase has a slow rate of catalysis: steady-state k(cat) values for the Escherichia coli, Magnaporthe grisea, and spinach enzymes are 0.024 , 0.052, and 0.023 s(-1), respectively, at pH 7.5 and 25 degrees C. Followi ng the formation of an imine connecting the two substrates 3,4-dihydroxy-2- butanone 4-phosphate and 4-ribitylamino-5-amino-2,6-dihydroxypyri dine, the re is a chemically difficult isomerization. Calculated estimates of the fre e energy barrier for the isomerization are equal to or greater than 15 kcal /mol at 25 degrees C. Free energies calculated from the steady-state k(cat) values at 25 degrees C for the E. coli, M. grisea, and spinach enzymes are 19.7, 19.2, and 19.7 kcal/mol, respectively. The single-turnover rate (pre -steady state) at pH 7.5 and 25 degrees C for the M. grisea enzyme is 140-f old greater than the steady-state rate and it has a free energy barrier of 16.3 kcal/mol. In the pre-steady state the M. grisea enzyme has a pK(a) of 5.8, plausibly reporting the proposed general base of catalysis (His127). T he M. grisea enzyme has an off rate of 0.37 s(-1) for its product, 6,7-dime thyl-8-ribityllumazine, approximately 7-fold higher than k(cat) and 20-fold lower than the single-turnover rate. The off rate for the product orthopho sphate is about 1 s(-1). Thus, for the M. grisea enzyme at pH 7.5 and 25 de grees C, product dissociation is significantly rate limiting to the steady- state rate of catalysis, whereas the isomerization step limits the single t urnover rate. The spinach and E. coil enzymes display a significant lag in pre-steady state, suggesting that substrate association is significantly ra te limiting for these catalysts. Temperature studies on the enzyme-catalyze d rates for the three enzymes indicate a dominating enthalpic term. (C) 200 0 Academic Press.