Triosephosphate Isomerase
File 2YPI
 
 
 
 
 
  This is the crystal structure of a triosephosephate isomerase molecule that is bound to a 2-phosphoglycolate (PGA) molecule.  Triosephosphate isomerase is an enzyme that catalyzes the interconversion of dihydroxy acetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP).  The PGA molecule acts as an inhibitor by binding to the active site of the enzyme and changing the conformation of the molecule.  PGA binds to the enzyme with an affinity more than 100 times that of either DHAP of GAP.  Due to this high binding affinity (as well as certain stereochemical qualities and charge configurations) it is believed that PGA is a transition state analogue.  PGA differs from the true transition state in that a methanol group is replaced by a single oxygen atom. Rotate the molecule to observe the PGA molecule in the binding pocket.  The selected residues form extensive hydrogen bonds with the PGA molecule.  The carboxylate oxygen #1 of PGA forms a hydrogen bond with the protenated carboxylate oxygen of glu165.  Carboxylate oxygen #2 of PGA forms a hydrogen bonds with lys12 and his95.  And the terminal phosphate oxygens of PGA form hydrogen bonds with the amide nitrogens of gly171 and ser211.
This glu165 becomes protenated upon binding of the PGA.  This is due to the presence of the negatively charged carboxylate of PGA in the active site.  The protenation of glu165 forms a very strong bond between the two carboxylates and prevents a destabilizing repulsion that otherwise would have occurred between gluís side chain and the anionic carboxylate of PGA.

Upon binding of either PGA or the substrate to the active site the enzyme undergoes a conformational change.

This 10 amino acid loop undergoes significant conformational changes upon binding of the substrate or PGA.  In the bound conformation the loop closes down over the substrate as seen here.  In the unbound conformation the loop shifts to an ìopenî conformation.  The loop forms many hydrogen bonds and Van-der Waals interactions with the PGA (or substrate).  The loop itself is essentially a rigid body and relative position of these ten amino acids to each other does not change during the shift from the open to the closed conformation.
It is thought that the closing of this loop around the substrate shields the active site from bulk water and prevents the phosphate elimination reaction form occurring.  In order for the phosphate elimination reaction to occur the bridging oxygen must develop a negative charge or be protenated.  By shielding the active site from the bulk solvent (which could potentially stabilize a negative charge on the oxygen) the enzyme makes the formation of a negative charge unfavorable and thus prevents this reaction from occurring.
 
 
Primary Citation:

Lolis, E., Petsko, G. A.: Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-A resolution: implications for catalysis.. Biochemistry 29 pp. 6619 (1990)
 

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