Squalene is a polyalkene, derived in plants and animals by a series of S_N2 reactions starting with isopentenyl phosphate.

It is oxidized by a monooxygenase enzyme, using O2 and NADH, to an oxirane.

The oxirane, in order to undergo cyclization, must adopt the conformation shown; we do not know whether this conformational change is enzyme-induced, or whether the enzyme simply binds this conformation selectively.

The enzyme then uses the CO₂H of an aspartic acid side chain to initiate an acid catalyzed ring opening, in which a neighboring double bond is the nucleophilic species:

As each carbocation is formed, it is attacked by another double bond, producing a cascade of steps:

The individual carbocation appear to exist as discrete intermediates, retained within the central cavity of the enzyme.

As diagrammed here the cyclization is the animal version; plants skip the oxidation step, and also cyclize through an all-chair conformation leading to a pentacyclic species:

The crystal structures that are available all are of the plant enzyme, but it is believe that the structures and catalytic functions are very similar. The two types of enzymes have about 25% sequence identity, both have several regions rich in aromatics, and both have aspartates as essential residues - about which more later.

The plant enzyme is suggested to be more primitive: that is to more nearly resemble the evolutionary ancestor of all the cyclases, for several reasons:

• It is anaerobic: it cyclizes squalene directly without oxidation, suggesting development prior to photosynthesis

• The reaction catalyzed is simpler, since it involves no skeletal rearrangement

• The plant enzyme is less specific than the one from eukaryotes; it will cyclize both stereoisomers of oxidosqualene as well as its natural substrate, whereas the eukaryotic enzyme will only cyclize oxidosqualene

A crystal structure is available for the plant enzyme, complexed with a competitive inhibitor (pdb1sqc):

The enzyme is composed of a large central cavity, the active site, situated between two helical domains. The active site is accessed through a nonpolar channel between the helices of the lower domain.

The channel is surrounded by a nonpolar "plateau", suggesting that this portion of the enzyme lies in the middle of the cell membrane.

Site-directed mutations indicate that both Asp376 and Asp377 are required for activity (that is, if we clone the enzyme with some other amino acid in place of either of these, the enzyme loses activity).

The double requirement may be related to the need to enhance the acidity of one carboxyl by hydrogen bonding it to the other. The graphic below displays the two aspartates in yellow:

Clearly, they are positioned so they could supply a proton to the double bond of the natural substrate.

The binding pocket is lined with aromatic residues to stabilize intermediate carbocations; I have colored in green a few Phe (365, 437, and 601) just to show this.