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 25Β°C. Following the formation of an imine connecting the two substrates 3,4-dihydroxy-2-butanone 4-phosphate and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, there is a chemically difficult isomerization. Calculated estimates of the free energy barrier for the isomerization are equal to or greater than 15 kcal/mol at 25Β°C. Free energies calculated from the steady-state k cat values at 25Β°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 (presteady state) at pH 7.5 and 25Β°C for the M. grisea enzyme is 140-fold 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). The M. grisea enzyme has an off rate of 0.37 s -1 for its product, 6,7-dimethyl-8-ribityllumazine, approximately 7-fold higher than k cat and 20-fold lower than the singleturnover rate. The off rate for the product orthophosphate is about 1 s -1 . Thus, for the M. grisea enzyme at pH 7.5 and 25Β°C, product dissociation is significantly rate limiting to the steady-state rate of catalysis, whereas the isomerization step limits the single turnover rate. The spinach and E. coli enzymes display a significant lag in pre-steady state, suggesting that substrate association is significantly rate limiting for these catalysts. Temperature studies on the enzyme-catalyzed rates for the three enzymes indicate a dominating enthalpic term.