Effects of temperature on enzyme activity and labilities of intermediates and cofactors involved in chimeric EM pathway. (A) The enzyme activities were determined under the standard assay conditions (HEPES-NaOH, pH 7.0) at indicated temperatures. Specific activities were given as those in the crude extract of recombinant E. coli having each enzyme. One unit was defined as the amount of enzyme catalyzing the consumption of 1 μmol of the substrate per minute. (B) The intermediates and cofactors were dissolved in 50 mM HEPES-NaOH (pH 7.0) and incubated for 1 h at 0, 40, 50, 60, and 70°C. The residual concentration of intermediates was determined by CE-TOFMS. ATP, ADP, NAD+, and NADH (0.1 mM) were determined as described in Materials and methods.