Recall that site-directed mutations indicate that both Asp376 and Asp377 are required for activity.

• The double requirement may be related to the need to enhance the acidity of one carboxyl by hydrogen bonding it to the other.

Squalene-Hopene Cyclase with Inhibitor

They are positioned so they could supply a proton to the double bond of the natural substrate. The identy of the residue that removes the proton at the very end of cyclization, producing hopene, is less certain.

Wendt, who did the crystal structure [Science, 1997, 277, 1811], suggest that it may be Trp169 (below, left), or perhaps a water molecule in a hydrogen bonded network with Gln262, Glu45 and Glu93, and Arg127 (which are not shown above).

The binding pocket is lined with aromatic residues to stabilize intermediate carbocation; I have picked out a few to show this.

Hoshino and Sato [Chem. Comm., 2002, 291] provide a nice graphic in which the squalene structure has been superposed on the residues surrounding the cavity:

The tightness of the pocket is notable in the graphic below, a slab 50% through a space-filling model:

• Except for the active ones noted above, no Glu or Asp residues lie along the sides of the pocket, although Hoshino and Sato include a stabilizing role for Asp377.
  • This could mean there are no carbocation intermediates needing stabilization

As noted before, the structures of the pro- and eukaryotic enzymes are quite similar, despite only about 25% sequence identity.

Human Oxidosqualene Cyclase (1w6k)	Human Oxidosqualene Cyclase (1w6k), Lanosterol Bound

Again, the conserved sequence in colored in violet.

The human enzyme is a monotopic membrane protein:

• It inserts into, but does not span, the membrane

• The enzyme tips to the right (as drawn above), inserting parts of two helices into the polar area of the membrane

• The catalytic site opens toward the membrane surface

Human Oxidosqualene Cyclase (1w6k), A/B Ring Interactions with Lanosterol
Human Oxidosqualene Cyclase (1w6k), C/D Ring Interactions with Lanosterol

• The acidity of the catalytic aspartate (455) is increased by H-bonding to Cys 533 and Cys 456

• Cation-p interactions with Trp367, Phe444, and Trp581 can stabilize the carbocation intermediates at C6 and C10

• Cation-p interactions with Phe696 and His232 can stabilize the C20 protosteryl cation

• His232 is the nearest basic residue that could deprotonate the final cation

In sum, with a few differences in specific residues for stabilization of cations and facilitation of protonation, the two types of enzyme operate by very similaar mechanisms.

Their differences lie in the fit of substrates into the binding pocket.

So, Does it all zip up at once? Violating the Fort Curly Arrow Rule? Current best guess is yes.

Oxidosqualene cyclase in now the target of a large effort in anticholesteric drugs; here is a structure of one bound:

Human Oxidosqualene Cyclase with Anticolesteric R71 (1w6j)

Clearly, this is classic competitive inhibition, just like the statins.