Biomedical Engineering Reference
In-Depth Information
10.4 Restoration of the Catalytic Triad by
Mutagenesis Does Not Rescue Protease Activity
Initial enzyme assays performed with SMIPP-Ss I1, G2, and B2 on methyl-
coumarin peptide substrates (AAPF, AAF, LLVY, LY, VKM, R, GGR, GPR,
and APA) revealed no proteolytic activity for wild-type proteins as well as for
mutants with their glycosylation sites removed (typically by mutation of Asn
residues to Gln).
In order to restore the catalytic triad as well as unblock the S1 subsite, two
mutants have been studied: Q41H/N85D/A186S/Y202A of the SMIPP-S-D1
mature sequence and the mutant A184S/Y200A of the SMIPP-S-I1 mature
sequence. Consistent with structural data, neither mutant displayed protease
activity on the range of substrates described above, suggesting that the absence
of protease activity is not solely due to these mutations and is most likely a
result of structural rearrangements in the oxyanion hole and S1 subsites, and at
least in the case of SMIPP-S-I1, these 'zymogen-like' characteristics appear to
play a role in the failure to reactivate SMIPPs by mutagenesis.
Taken together with the structural data, these findings suggest that SMIPPs
have diverged significantly from the ancestral serine protease, resulting in an
irreversible culmination of mutations in and around the active site that result in
extensive structural rearrangements, effectively occluding the binding of sub-
strates (Figures 10.1 and 10.2).
10.5 Phylogenetic Analysis of the SMIPP Family
A codon-based alignment of the SMIPP sequences was divided, with reference
to the crystal structure of SMIPP-S-I1, into three functionally distinct regions:
surface exposed active site residues; surface-exposed residues outside the active
site; and buried residues. The rates of synonymous mutation (a polymorphism
yielding an identical amino acid) versus non-synonymous mutation (causing an
amino acid substitution) were inferred and are shown in Table 10.1. The values
reveal that surface residues outside the active site are subject to significant
(p
o
0.001) purifying (negative) selection, suggesting that evolutionary pressure
is operating to maintain the functional and/or structural integrity of the
SMIPPs as a whole. In contrast, residues within the active site appear to be
undergoing neutral drift and gradual diversification. This indicates that, on
Table 10.1 Differences between estimated d
S
and d
N
values for different
structural regions of SMIPPs.
d
S
d
N
Active site, solvent exposed
1.22
1.21
0.73
**
External to active site, solvent exposed
1.72
0.47
**
Buried
1.48
**
Significant difference (p
o
0.001) between d
S
and d
N
values.
