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note
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As previously reported, the activity of P. roqueforti aristolochene synthase is a simple linear function of protein concentration only within a specific concentration range.10 The activity of wild-type aristolochene synthase was linearly proportional to protein concentration only up to 20 nM, after which the measured activity per mg protein decreased, with negligible increase in total activity above 60 nM protein (Figure 2A, Table 1). Similar behavior was also observed for the A. terreus aristolochene synthase, which displayed nonlinear dependence on protein concentration above 27 nM protein. To allow meaningful comparisons of steady state kinetic parameters, therefore, we first determined the concentration dependence of aristolochene synthase activity for all mutants prepared in this study. (Table 1) Kinetic assays of each mutant were then carried out only at protein concentrations within the experimentally established linear range.
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The activity of the Y92F mutant was found to be a simple linear function of protein concentration only up to 50 nM (Figure 2B, Table 1), with only minor increases in total cyclase activity above 80 nM mutant protein.
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m compared to the wild-type. The reported steady-state kinetic parameters were unfortunately determined at concentrations well above what turns out to be the maximum 50 nM protein concentration for linear dependence of Y92F activity (cf Table 1), thereby compromising their quantitative significance. On the basis of an observed increase in the proportion of germacrene A (28%), of unspecified configuration, the implausible suggestion was made that Tyr-92 must be the normal active site Lewis acid responsible for the cyclization of the germacrene A intermediate, although the major product isolated was still aristolochene. These workers also reported the isolation of the coproducts α-selinene (6), β-selinene, and selina-4,11-diene, none of which are detectable in our analysis of the same Y92F mutant (Table 2). The latter compounds are well-known artifacts of the acid-catalyzed rearrangement of germacrene A. Rearrangement of germacrene A is easily avoided by chromatography over aluminum oxide instead of silica gel and the use of sodium sulfate in place of magnesium sulfate as drying agent.
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