Transcript Lecture 19 - University of Wisconsin–Madison

Chemical mechanism is dominant
• Nature selects the protein for divergent evolution from a
pool of enzymes whose mechanism provide a partial
mechanism, or provide the means to stabilize an
energetically unfavorable intermediate or transition state.
• The original enzyme might have acquired a low level of
the new activity through adventitious mutations.
Subsequently the new enzyme would evolve (selective
pressure) to increase the proficiency of the new reaction
at the loss of its original.
• The old and new enzymes would be homologous and
belong to a mechanistically diverse superfamily.
• Examples of this type of evolution are seen in the enolase,
amidohydrolase/phosphotriesterase, and crotonase
superfamilies.
Structural Alignment of Phosphoribosylanthranilate Isomerase,
Indoleglycerol Phosphate Synthase, and TrpS(a)
A
B
Structural alignments of (a) PRAI (green) and IGPS (purple) and (b) IGPS
(purple) and a-TrpS (yellow). The ligand for the PRAI reaction is
shown in yellow, the ligand for the IGPS reaction is shown in orange,
and the ligand for the a-TrpS reaction is shown in blue.
Superposition of OMPDC and KGPDC
Structural alignment of 3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC)
(purple) with bound L-gulonate 6-phosphate (yellow) and OMPDC (green) with
bound UMP (orange). Although they share limited sequence identity, both enzymes
adopt a conserved (a)8 barrel fold. The quaternary relationship between the two
individual subunits in the dimer is highly conserved.
Location of Catalytic groups in the Super Family
Strand
1
2
Enolase
3
4
5
6
7
8
D
E
DE
K
(H)
(K)
H
E
MR
KxK
D
EEP
E
D
MLE
KxK
D
EQP
DE
K
(E)
Base blue, acid red, and Mg2+ ligands green.
A striking feature of this super family is the conserved location of the
catalytic groups. This supports the hypothesis that these enzymes arose
by divergent evolution, since there is no reason that the functional groups
should lie on the same strand.
The relative orientation (order) of the groups around the barrel is required
to provide adequate coordination of the metal and to place the catalytic
bases on opposite sides of the substrate. There is no strict structural
requirement that the metal ligands lie on the third, fourth, and fifth strands.
OMPDC and KGPDC Use the Same Active Site
Residues for Different Purposes
The active site resides in
OMPDC and KGPDC are
remarkably similar, yet they
serve different purposes in
their reaction schemes.
Conclusions
• It seems likely that in one case the (/a)8 barrel arose via
gene duplication of a smaller folding domain.
• Did all (/a)8 barrels arise with this mechanism? Probably
not.
• Structures provide insight into enzyme evolution, but not
always answers!