Synthetic Efficiency in Enzyme Mechanisms Involving Carbocations: Aristolochene Synthase

An intramolecular proton-transfer mechanism has been proposed for the carbocationic cyclization\nof farnesyl pyrophosphate (FPP) to (+)-aristolochene catalyzed by aristolochene synthase. This novel\nmechanism, which is based on results obtained by high-level <i>ab initio</i> molecular orbital and density functional\ntheory calculations, differs from the previous proposal in the key step of carbocation propagation prior to\nthe formation of the bicyclic carbon skeleton. Previously, germacrene A was proposed to be generated as\nan intermediate by deprotonation of germacryl cation followed by reprotonation of the C6−C7 double bond\nto yield eudesmane cation. In the mechanism proposed here the direct intramolecular proton transfer has\na computed barrier of about 22 kcal/mol, which is further lowered to 16−20 kcal/mol by aristolochene\nsynthase. An alternative pathway is also possible through a proton shuttle via a pyrophosphate-bound\nwater molecule. The mechanism proposed here is consistent with the observation that germacrene A is\nnot a substrate of aristolochene synthase. Furthermore, the modeled substrate−enzyme complex suggests\nthat Trp 334 and Phe 178 play key roles in positioning the substrate in the reactive orientation in the binding\npocket. This is consistent with experimental findings that mutations of either residue lead to pronounced\ngeneration of aborted cyclization products.