Prenyltransferases substrate binding

Although several previous reports claimed that the enzyme had been purified, Gebler and Poulter (1992) appear to have been the first to fully characterize the activity of the purified DMAT synthase. The enzyme was purified from Claviceps fusiformis ATCC 26245 [erroneously annotated in type specimen collections as a C. purpurea strain (Pazoutova and Tudzynski, 1999)]. The monomeric size was estimated at 53 kDa, and by gel filtration analysis the native enzyme was determined (at 105 kDa) to be a homodimer. Unlike other prenyltransferases, no metal ion requirement has been noted. However, when assayed in a buffer with 4 mM Ca2+, the purified protein gave a specific activity of 500 nmol/min/mg, essentially the same as with 4 mM Mg2+, but approximately twice that of the measured without added divalent cations and with the chelator EDTA included in the assay buffer. These divalent metal cations eliminated negative cooperativity of substrate binding observed both for dimethylallyl diphosphate and L-tryptophan, indicating that Ca2+ and Mg2+ probably had allosteric effects. In buffer with 4 mM MgCl2 the KM for dimethylallyl diphosphate was 8 jlM, and the KM for L-tryptophan was 12 xM. The enzyme product was authenticated by mass spectrometry, UV spectrometry, and -NMR. 

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