Figure 1.
Proposed glycosyltransferase mechanisms.
(A)A double displacement mechanism utilizing two inversions with net retention of stereochemistry involving a covalent glycosyl-enzyme intermediate. The individual steps are inverting via (B) an SN2 process. Inverting Leloir glycosyltransferases promote a backside nucleophilic attack on C1 by the acceptor from an inline (usually equatorial) position, with resulting inversion of the anomeric bond stereochemistry. Alternative mechanisms for retaining glycosyltransferases include: (C) an orthogonal mechanism consisting of nucleophilic attack on C1 by the acceptor concurrent with leaving group loss from a position approximately at right angles to the C1-leaving group axis; (D) an SNi mechanism involving an intermediate with oxocarbenium character followed by rapid internal nucleophilic attach by the acceptor nucleophile; or (E) an SN1 mechanism involving a discreet oxocarbenium intermediate. All mechanisms require proton transfers of the hydroxyl hydrogen of the acceptor to an enzymatic baseor the departing leaving group.
Table 1.
GT-A fold glycosyltransferase families with deposited structures.
Figure 2.
The a-f nomenclature used to describe octahedral binding partners.
Inverting enzymes such as GalT1 (top) achieve nearly perfect octahedral geometry about the coordinated metal ion (displayed angles of 81° and 91° compared to ideal octahedral 90° bond angles) with subsequent “inline” (approaching 180°) placement of the acceptor nucleophile for classic inverting SN2 backside attack. Retaining enzymes such as GTA (bottom), however, use an arrangement of acidic residues, often with acute bidentate Asp coordination, which severely skews metal geometry (displayed angles of 54° and 115°) and allots sufficient room between phosphate oxygens for orthogonal attack from the acceptor. U is uridine, C1 is donor galactose C1.
Figure 3.
Opposed to the placement of the acceptor nucleophile (Green spheres), the closest polar residues to leaving group β-phosphate O3 and C1 lay acutely (67° and 75°, respectively) for inverting enzymes (A,B) and lie nearly in-line (171° and 155°, respectively) for retaining enzymes GTA (C,D). This may help to stabilize the associative intermediates without hindering the opposite angle of attack from the acceptor molecule nucleophile. Also, the O3– C1vectors lay looselyperpendicular to the enzyme macrodipole vectors to stabilize the inverting transition states (green arrows) (A,B), and loosely parallel to stabilize the retaining transition states (C,D) (green ⊕, dipole oriented with the cationic end above the page and the anionic end in the page).
Table 2.
Active site residue identities and geometric values.
Figure 4.
Retaining and inverting enzymes are entirely orthogonal.
Theβ-sheets of the metal-nucleotide-sugar binding GT-A foldsof glycosyltransferase structures are superimposed by centering on the metal ion (magenta sphere) and the coordinated phosphates reveal that the general architecture of entire inverting or retaining enzymes are skewed by ∼90°. Color coding: purple, inverting GlcAT-I; blue, inverting GalT1; red, retaining GTA; pink, retaining LgtC. Left panel shows the superimposed solvent-accessible surfaces of the four structures with the folds embedded; the right panels isolate the β-sheets and show orthogonal perspectives.
Figure 5.
Geometries and energetics of mechanisms.
(left) More O'Ferrall-Jencks plot illustrating the concurrent reaction coordinate geometry changes of proposed mechanisms. (right) Comparative reaction profile diagrams for dissociative (SN1, SNi) and orthogonal pathways. The relative energetics and rate-limiting transition state locations of the three pathways are speculative and are offset for clarity, but both SN1 and SNi displacement would certainly involve an intermediate in anenergy well whereas the orthogonal mechanism does not.