FIGURE 1.

Triokinase and cyclizing lyase activities of hTKFC. A, coexpression of GA kinase with DHA kinase and FMN cyclase activities in BL21 cells transformed with plasmid pGEX-6P-3-hF2 that encodes a GST-human DHA kinase/FMN cyclase fusion protein (24). The lysate supernatant was chromatographed on a GSH-Sepharose column to which the GST-labeled protein adsorbed. The recombinant protein was eluted from the column after an overnight incubation with PreScission protease. In the fractions collected, the three enzyme activities shown were assayed, and the presence of the recombinant 60-kDa protein band composed of the 575 amino acids of human DHA kinase/FMN cyclase plus an N-terminal extension of 11 amino acids left by the cut with PreScission was detected by SDS-PAGE. B, substrate specificity of the kinase and cyclizing lyase activities. Activity with S.D. (error bars) is the mean of three experiments, each performed with two different amounts of enzyme evaluated either by continuous spectrophotometric recording (kinase) or by HPLC at three time points (cyclizing lyase) under conditions of linear response with respect to incubation time and hTKFC amount. Kinase activities were assayed with 0.5 mm phosphoryl acceptor, 5 mm (deoxy)nucleoside triphosphate donor, and 10 mm MgCl₂. Cyclizing lyase activities were assayed with 0.5 mm substrate and 6 mm MnCl₂. Ap2A, diadenosine pyrophosphate.