close proximity, while nonproductive movements are restricted. This rateenhancing entropy effect is very similar to an intramolecular reaction, where the
reacting groups are prearranged in close proximity. In contrast, intermolecular
reactions are slower because the reacting partners have to find each other through
diffusion [123–127]. In many proteins, the substrate has to approach the active site
through a tunnel, which ensures its proper orientation, which enhances the number
of productive catalytic events. In contrast, small chemical catalysts need many
more collisions for successful catalysis.
Electrostatic and Covalent Catalysis
Most transition states involve charged intermediates, which are stabilized within the
active site of an enzyme via ionic bonds in ‘pockets’ or ‘holes’ bearing a matching
opposite charge. Such charges are provided by (Lewis acid-type) metal ions, typically
Zn
2+ or are located on acidic or basic amino acid side chains (such as His, Lys, Arg,
Asp, or Glu). It is important to note that the (modest) pK a of typical amino acid side
chains, such as –NH 3
+ or –CO 2
À can be substantially altered up to 2–3 pK a -units
through neighboring groups within the enzyme environment. As a consequence, the
(approximately neutral) imidazole moiety of His can act as strong acid or base,
depending on its molecular environment. Computer simulation studies suggested
that in enzymes electrostatic effects provide the largest contribution to catalysis
[128]. As a prominent example, the tetrahedral intermediate of carboxyl ester hydrolysis is stabilized in serine hydrolases by the so-called ‘oxyanion hole’ (Scheme 2.1).
Many enzymes form covalent bonds with their substrates during catalysis, such
as the acyl-enzyme intermediate in carboxyl ester hydrolysis (Scheme 2.1) or the
glycol monoester intermediate in epoxide hydrolysis (Scheme 2.84). Despite the
covalent enzyme-substrate bond, such species are metastable and should be
regarded as ‘activated intermediates’. Some enzymes utilize cofactors, such as
pyridoxal phosphate (PLP) or thiamine diphosphate (TPP), to form covalent intermediates during catalysis.
Since some enzymes can act faster than what would be predicted by the ‘overthe-barrier’ transition-state model (ΔΔG
6 ¼ ), ‘through-the-barrier’ quantum tunneling of protons or electrons has been postulated [129, 130].
Three-Point Attachment Rule
This rationale to explain the enantioselectivity of enzymes was suggested by
A.G. Ogston [131]. Since chirality is a quality of space, a substrate must be
positioned firmly in three dimensions within the active site of an enzyme in order
to ensure spatial recognition and to achieve a high degree of enantioselection. As a
consequence, at least three different points of attachment of the substrate onto the
active site are required.
19
Although the majority of chiral molecules subjected to biotransformations
possess central chirality located on an sp
3 -carbon atom, all types of chiral molecules can be ‘recognized’, including compounds bearing a stereogenic sp
3 -
19 The following rationale was adapted from [132].
16
1 Introduction and Background Information
reacting groups are prearranged in close proximity. In contrast, intermolecular
reactions are slower because the reacting partners have to find each other through
diffusion [123–127]. In many proteins, the substrate has to approach the active site
through a tunnel, which ensures its proper orientation, which enhances the number
of productive catalytic events. In contrast, small chemical catalysts need many
more collisions for successful catalysis.
Electrostatic and Covalent Catalysis
Most transition states involve charged intermediates, which are stabilized within the
active site of an enzyme via ionic bonds in ‘pockets’ or ‘holes’ bearing a matching
opposite charge. Such charges are provided by (Lewis acid-type) metal ions, typically
Zn
2+ or are located on acidic or basic amino acid side chains (such as His, Lys, Arg,
Asp, or Glu). It is important to note that the (modest) pK a of typical amino acid side
chains, such as –NH 3
+ or –CO 2
À can be substantially altered up to 2–3 pK a -units
through neighboring groups within the enzyme environment. As a consequence, the
(approximately neutral) imidazole moiety of His can act as strong acid or base,
depending on its molecular environment. Computer simulation studies suggested
that in enzymes electrostatic effects provide the largest contribution to catalysis
[128]. As a prominent example, the tetrahedral intermediate of carboxyl ester hydrolysis is stabilized in serine hydrolases by the so-called ‘oxyanion hole’ (Scheme 2.1).
Many enzymes form covalent bonds with their substrates during catalysis, such
as the acyl-enzyme intermediate in carboxyl ester hydrolysis (Scheme 2.1) or the
glycol monoester intermediate in epoxide hydrolysis (Scheme 2.84). Despite the
covalent enzyme-substrate bond, such species are metastable and should be
regarded as ‘activated intermediates’. Some enzymes utilize cofactors, such as
pyridoxal phosphate (PLP) or thiamine diphosphate (TPP), to form covalent intermediates during catalysis.
Since some enzymes can act faster than what would be predicted by the ‘overthe-barrier’ transition-state model (ΔΔG
6 ¼ ), ‘through-the-barrier’ quantum tunneling of protons or electrons has been postulated [129, 130].
Three-Point Attachment Rule
This rationale to explain the enantioselectivity of enzymes was suggested by
A.G. Ogston [131]. Since chirality is a quality of space, a substrate must be
positioned firmly in three dimensions within the active site of an enzyme in order
to ensure spatial recognition and to achieve a high degree of enantioselection. As a
consequence, at least three different points of attachment of the substrate onto the
active site are required.
19
Although the majority of chiral molecules subjected to biotransformations
possess central chirality located on an sp
3 -carbon atom, all types of chiral molecules can be ‘recognized’, including compounds bearing a stereogenic sp
3 -
19 The following rationale was adapted from [132].
16
1 Introduction and Background Information