User:Andrea Bauer/Sandbox 956

Short Introduction

Description

IIsoprene Synthase is supposed to be a dimeric enzyme which consists of 595 amino acids and has a molecular mass of 68,386 Da. The protein is made up of alpha-helices which form two alpha-helical domains. The N-terminal domain of the protein chain is folded similar to class II terpenoid synthases that are made up of (𝛼𝛼)6 barrels [Wendt et al.,1998]. Up to now there is no catalytic activity known for this domain. Quite the contrary regarding the C-terminal domain of the isoprene synthase: This domain shows up an 𝛼-helical class I terpenoid synthase fold and contains the active site which is surrounded by five 𝛼-helices. The active site of the enzyme is located in a deep hydrophobic pocket which ensures a protection of the reaction intermediate from water.

Catalyzed Reaction

The Isoprene Synthase catalyses the production of isoprene from the substrate dimethylallyl-diphosphate (DMAPP). During the reaction inorganic pyrophosphate is eliminated leading to the reaction products isoprene and inorganic pyrophosphate. The release of the pyrophosphate group leads to the generation of an allylic carbocation which is typical of class I terpenoid synthases [Wendt et al., 1998]. The occuring elimination mechanism is syn-periplanar and the leaving diphpsphate group acts as general base. The characteristic DDXXD-sequence motif of class I terpenoid synthases that binds to the diphosphate leaving group via Mg2+-ions facilitates the release of the pyrophosphate group.

Structure

The hydrophobic active site pocket has a higher affinity towards a 5-carbon substrate rather than to a 10-carbon complex and Van der Waals interactions take place with DMASPP and the isoprenoid moiety of the active site on F338, V341 and F485. PcISPS remains in the open conformation while being in the DMASPP complex. It was suggested that the diphosphate leaving group itself serves as the general base. So a syn-periplanar elimination reaction was suggested with the development of an intermediate carbocation, which lead to the assumption that the isoprene generation is catalyzed by a substrate-assisted mechanism. PcISPS was found to be monomeric in crystallization, but it would be unusual to exhibit positive cooperativety for a monomeric enzyme like it was found to show up. There was evidence, that this cooperativity results from dimeric quarternary structure, where C-terminal catalytic domains interact to form an isologous dimer.

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