Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity

Article abstract of DOI:10.1016/S0968-0896(99)00320-X

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S₁ pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive in

Full text of DOI:10.1016/S0968-0896(99)00320-X

Bioorganic & Medicinal Chemistry 8 (2000) 563±570
Benzophenone Boronic Acid Photoanity Labeling of Subtilisin
CMMs to Probe Altered Speci®city
Grace DeSantis, a Christian Paech b and J. Bryan Jones a,*
aDepartment of Chemistry, University of Toronto, 80 St George Street, Toronto, Ontario, M5S 3H6, Canada
bGenencor International Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA
Received 21 September 1999; accepted 27 October 1999
AbstractÐA transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the di€erences in
the topology of the S1 pocket of chemically modi®ed mutant enzymes (CMMs). The BBP proved to be an e€ective competitive
inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which
were chemically modi®ed with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue.
As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large
hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the
charged CMMs, S166C-S-CH2CH2SO3 or S166C-S-CH2CH2NH+3 , was reduced 170-fold and 4-fold respectively. Photolysis of the
WT-SBL±BBP enzyme±inhibitor (EI) complex, inactivated the enzyme and e€ected the formation of a covalent crosslink between WT
and BBP. The crosslink was identi®ed at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1
hydrophobic pocket of SBL and its modi®cation thus established binding of the benzophenone moiety in S1. Photolysis of the EI
complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3 , or S166C-S-CH2CH2NH+3 and BBP under the same conditions did not
inactivate these enzymes, nor e€ect the formation of a crosslink. These results corroborated the kinetic evidence that the active site
topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11±BBP EI
complex only inactivated 50% of the enzyme after 12 h, it still e€ected the formation of a covalent crosslink between the CMM and
BBP, again at Gly127. However, this photolytic reaction was less ecient than with WT, demonstrating that the S1 pocket of S166C-
CH2-c-C6H11 is signi®cantly restricted compared to WT, but not as completely as for the other CMMs. # 2000 Elsevier Science
Ltd. All rights reserved.
Introduction
introduction of a cysteine residue at a key active site
position via site-directed mutagenesis whose side chain
is then thioalkylated with an alkyl methanethiosulfonate
reagent (1a±d) to yield a chemically modi®ed mutant
enzyme (CMM) of the serine protease subtilisin Bacillus
lentus (SBL).10±14
Enzymes are widely exploited as catalysts in organic
synthesis due to their regio-, stereo-, and enantio-selec-
tivity under very mild reaction conditions.1±3 In order to
expand the synthetic utility of enzymes even further,
additional insights into the precise nature of the factors
which control their structural and stereospeci®city are
needed.4,5 Consequently, the creation of enzymes with
new catalytic activities, and tailoring the speci®city of
existing ones to accommodate unnatural substrates has
become an area of current intense interest.6
Mutagenesis to cysteine and chemical modi®cation of
the S1 pocket Ser166 residue of SBL was chosen for this
study. Previously, the S1 binding pocket properties of the
S166C CMMs of SBL were probed using a series of
boronic acid transition state analogue competitive inhi-
bitors.9,15 The inhibitor probing approach revealed sub-
stantial changes in the S1 binding pocket and also yielded
insights into the molecular basis for these changes.15
Since X-ray crystal structures of the boronic acid inhi-
bitors bound to this and other CMMs are not available,
we considered the strategy of employing an active site
directed photoanity label to map SBL's binding site in
order to further investigate the altered binding modes of
boronic acid inhibitors to the S1 pocket.
Recently, the combined chemical modi®cation±site-
directed mutagenesis approach has emerged as an e-
cient method to tailor and probe enzyme speci®city.7±9
This strategy is outlined in Scheme 1, and entails the
*Corresponding author. Tel.: +1-416-978-3589; fax: +1-416-978-1553;
e-mail: jbjones@alchemy.chem.utoronto.ca
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00320-X

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G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
Preparation of p-boronic acid benzophenone
The p-boronic acid benzophenone (BBP, 1) photoprobe
designed for the current study was prepared as outlined
in Scheme 2. Brie¯y, 4-bromobenzophenone (3) was pro-
tected as the glycal in re¯uxing benzene.36±37 The glycal (4)
was then subjected to halogen±lithium exchange by reac-
tion with n-butyl lithium, followed by treatment with
trimethyl borate to generate the corresponding dimethyl
borate.31,37 To facilitate puri®cation,31 the dimethyl
borate was subsequently deprotected in sulfuric acid
and reprotected with ethylene glycol, generating the
diglycal (5), which was recrystallized from benzene. The
puri®ed diglycal (5) was then fully deprotected in
re¯uxing 6 M HCl,37 yielding the p-boronic acid benzo-
phenone (BBP, 1) in 77% overall yield (Scheme 2).
Scheme 1.
Active site directed photoanity labels16±20 have been
employed extensively to probe enzyme active sites,21,22
and to provide structural information complementary
to that which may be available from X-ray crystal-
lography23 or NMR24,25 approaches. Benzophenone
(BP) inhibitor21,22 and substrate26 derivatives have
been employed extensively in active-site directed photo-
anity labeling studies.17,18±27 For the current study,
the photolabile p-boronic acid benzophenone (BBP, 1)
was designed to incorporate the boronic acid transi-
tion state analogue inhibitor structure.28,29±34 The
BBP photoprobe has the active site-directing phenyl
boronic acid inhibitor incorporated into the structure
of BP rather than tethered to the end of the photo-
probe, thereby reducing nonspeci®c labeling com-
plications.18
Evaluation of competitive inhibition by BBP
Initially, the ecacy of binding of the designed photo-
labile competitive inhibitor, BBP, to each of the WT,
S166C-S-CH2C6H5, S166C-S-CH2-c-C6H11, S166C-S-
CH2CH2NH+3 and S166C-S-CH2CH2SO3 enzymes was
determined by measuring the catalytic activity using
suc-AAPF-pNA as substrate.38 The kinetic data are
summarized in Table 1 and, as expected, in all cases
BBP binds best to WT. BBP binding to S166C-
SCH2C6H5 (-a) and S166C-CH2-c-C6H11 (-b), with their
large hydrophobic side chains, is reduced by 86-fold
and 9-fold, respectively, compared to WT. Previously,
The binding pocket topographies of four representative
S166C CMMs and of the WT-SBL were investigated.
Two of these, S166C-S-CH2C6H5 (-a) and S166C-S-
CH2-c-C6H11 (-b) have a sterically constricted S1 pocket
while S166C-S-CH2CH2SO3 (-c) and S166C-S-CH2
+
CH2NH3 (-d) have a negatively and a positively
Scheme 2.
charged S1 pocket respectively (Scheme 1). In this
study we report the synthesis of the BBP reagent, its
evaluation as a competitive reversible inhibitor of WT-
SBL and of S166C CMMs (Scheme 1), and photolysis
of the corresponding BBP±EI complexes.
Table 1. Kinetic and inhibition constants for S166C CMMs with BBP
1
Enzyme
KM mM
kcat
s
kcat/KM
1
KI mMb
1a
mM
s
WT
0.55‹0.06
0.74‹0.09
0.61‹0.04
0.70‹0.06
0.60‹0.06
48‹2
6.9‹0.4
8.7‹0.2
3.8‹0.1
16.3‹0.5
87‹10
9‹1
14.3‹0.9
5.4‹0.5
27‹3
0.037‹0.004
3.2‹0.04
0.32‹0.02
6.3‹0.6
S166C-S-a
S166C-S-b
S166C-S-c
S166C-S-d
Results and Discussion
Preparation of CMMs
0.15‹0.02
aMichaelis±Menten constants were measured by the initial-rate
method in 0.1 M phosphate bu€er (pH 7.5, 0.5 M NaCl) at 25 ꢀC with
suc-AAPF-pNA as the substrate.
bInhibition constants were determined under the same conditions by
the method of Waley.39
Each of the chemically modi®ed mutant enzymes
(CMMs) was prepared9 and characterized9,35 by the gen-
eral method described previously, yielding S166C-S-a to
-d as outlined in Scheme 1.

G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
Table 2. ESMS data for BBP treated WT-SBL
565
all the boronic acids evaluated15 exhibited poorer bind-
ing with the S166C-S-CH2C6H5 CMM compared to
WT, and therefore the increased KI of BBP with this
CMM is not unexpected. That BBP binding to S166C-
S-CH2-c-C6H11 is better than to S166C-S-CH2C6H5
may be due to the greater hydrophobicity of its side chain
or to di€erences in side chain orientation. BBP binding to
the charged CMMs, S166C-S-CH2CH2SO3 (-c) and
S166C-S-CH2 CH2NH+3 (-d), is reduced 170-fold and 4-
fold respectively compared to WT. This inhibition pattern
is comparable to that revealed by the previous boronic
acid inhibitor screens, for which all except for the
smallest inhibitor, phenyl boronic acid, exhibited poorer
binding to S166C-CH2CH2SO3 compared to WT.15 In
contrast, the apparently more accommodating nature of
the S1 pocket of S166C-CH2CH2NH+3 is in accord with
the fact that three of the ®ve boronic acid inhibitors
evaluated exhibited improved binding compared to
WT.15 Thus, BBP binding to SBL exhibits the same
activity patterns as did the previously evaluated boronic
acid transition state analogue inhibitors.
Treatment
Average mass Average mass
calculated
Interpretation
found
hv
26698
26888
26888
26698
26698
26702
26888
26891
26702
26706
WT enzyme
WT+BBP 2H2O
WT+BBP 2H2O
WT enzyme
2Â BBP+hv
10Â BBP+hv
2Â BBP+dark
10Â BBP+dark
WT enzyme
Figure 1. Time course for photolysis (350 nm) of SBL-WT and BBP.
Photolysis of enzyme±BBP complexes
The advantages of the BP photophore have been docu-
mented and include its chemical stability, its stability to
ambient light and its 330±360 nm photoactivation
range, which is not damaging to proteins.18 In addition,
BP is particularly well suited to the current probing
study since SBL's active site is quite hydrophobic and
BP exhibits preferential labeling of hydrophobic binding
regions in proteins.18 The photolysis of benzophenone
derivatives at l 320±360 nm e€ects an n to p* transition
which induces formation of a covalent link to amino
acid residues, or other functionalities, by H-abstraction
and radical recombination.17,18±27
the enzyme with BBP in the dark. However, for the WT
enzyme photolyzed in the presence of either a 2-fold or a
10-fold molar excess of BBP a mass increase consistent
with covalent linkage of BBP followed by the loss of
two moles of water, was observed, as summarized in
Table 2.
To identify the location of the crosslink of BBP to the
enzyme, peptide mapping analysis was employed.40
Speci®cally, each of the enzyme samples was denatured
®rst by acid then by urea and then degraded by trypsin
into fragments designated 1T to 13T, which correspond
to the tryptic fragments numbered sequentially from the
amino terminus of the enzyme. These fragments were
then separated by reverse-phase HPLC which, coupled
to mass spectrophotometric analysis, permitted the
identi®cation of each of the tryptic fragments on a pep-
tide map.40 Comparison of the mass and retention time
of the BBP-treated and photolyzed enzyme to that of
the control was used to identify the BBP modi®ed pep-
tide fragment. This revealed a modi®cation on tryptic
fragment 6T, which was comprised of amino acids 93±
143 in the linear sequence. Modi®cation resulted in the
disappearance of fragment 6T and yielded two new
peaks, a minor peak designated as 6T0 and a major peak
designated as 6T00. Both of these were more hydro-
phobic than 6T itself, as evidenced by their longer
retention times on reverse-phase HPLC, and showed
mass increases over 6T of 197 and 189 Da for 6T0 and
6T00, respectively (Fig. 2).
The initial photolysis time-course experiments were
done with WT-SBL. The photolysis reactions were con-
ducted at 4 ꢀC in slightly acidic bu€er (pH 5.8) in order
to reduce autolysis of the enzyme. The BBP photoprobe
was used at both a 2-fold and a 10-fold molar excess
concentration with respect to the enzyme. Control and
blank reactions were conducted under the same condi-
tions. Aliquots were withdrawn and speci®c activity
measurements made.
The photolysis time-course experiment revealed a time
dependent decrease in speci®c activity falling o€ to zero
after 60 min (Fig. 1). Both the 2-fold and 10-fold molar
excess of BBP reactions e€ected the same ®nal activity,
with the more concentrated reaction mixture resulting in
a slightly faster activity loss. For both the WT photo-
lyzed control (WT+hv) and the dark control (WT+
BBP) reactions, activity was unaltered. This established
that the activity losses observed were due exclusively
to the photochemical reaction between the enzyme
and BBP. After photolysis, each sample was puri®ed
and analyzed by ESMS. Both the WT photolyzed
(WT+hv) control and the dark (WT+BBP+dark)
control retained the original WT mass, demonstrating
that no chemical changes were induced by photolysis
of the enzyme in the absence of BBP nor by treatment of
The peptides 6T0 and 6T00 corresponded to the BBP
modi®ed tryptic fragment 6T and were isolated by
HPLC. The speci®c location of the covalent bond
between BBP and the 6T0 and 6T00 tryptic fragments
were identi®ed by automated amino acid sequencing of
the isolated peptides. For the minor peptide 6T0 the
Edman sequencing signal became weak after cycle 33

566
G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
revealing the sequence VLGASGSGSVSSIAQGLE-
WAGNNGMHVANLS126L127 128xxx (BPN0 number-
ing). However, it was not clear if the cycle stopped at this
point, or just weakened. In contrast, for the major peptide
6T00 an abrupt termination after cycle 32 was noted,
revealing the sequence . . .VANLS126 127xxx (BPN0 num-
L
crosslinking mechanism which accounts for this mod-
i®cation, and for the loss of two water molecules, and
which is consistent with the observed mass change, is
presented in Figure 3. The modi®cation site of the
minor peptide fragment 6T0 is not conclusive but may
be at residue Ser128.
G
bering). The sequence information obtained for the major
peptide 6T00 identi®ed a modi®cation at residue Gly127
(BPN0 numbering). This is highly signi®cant since Gly127
is located on the back wall of the S1 pocket. A proposed
The above results demonstrate the validity of the
approach for probing the active site of WT-SBL. Accord-
ingly, the same protocol was applied to the CMMs
S166C-S-a to -d, using a 10-fold molar excess of BBP, in
order to probe BBP binding changes. However, despite
a 12 h photolysis time, speci®c activity was not
decreased to zero for any of the CMM samples. None-
theless, each of the photolyzed CMMs and the dark
controls, were puri®ed and analyzed by ESMS. For
each of S166C-S-a, -c, -d, no modi®cation was indicated
by ESMS (Table 3). However, a mass increase of 220
Da was observed for S166C-S-CH2-c-C6H11 (-b)
10ÂBBP+hv, which is consistent (‹6 Da) with the
formation of a covalent bond between S166C-S-b and
BBP (calculated mass of BBP 226 Da).
Table 3. ESMS data for 10ÂBBP treated S166C-S-x
S166C-S-x/
treatment
Average mass Average mass
found
Interpretation
calculated
-a BBP+dark
-a BBP+hv
-b BBP+dark
-b BBP+hv
-c BBP+dark
-c BBP+hv
-d BBP+dark
-d BBP+hv
26836
27062
26842
27068
26853
27079
26790
27016
26847
26839
26844
27062a
26868
26867
26818
26818
Unchanged CMM
Unchanged CMM
Unchanged CMM
CMM+BBPa
Unchanged CMM
Unchanged CMM
Unchanged CMM
Unchanged CMM
Figure 2. HPLC-trace of (a) native and (b) BBP crosslinked WT-SBL:
tryptic fragments 1TÐ13T are identi®ed.
aPoorly resolved spectrum.
Figure 3. Proposed mechanism of BBP crosslinking: the photoactivated crosslinking of BBP to Gly127 and dehydration. The last dehydration step
forming a boronate ester between BBP and the enzyme may precede crosslinking.

G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
567
For the S166C-S-c-C6H11 CMM, both the dark control
Experimental
(S166C-S-b 10ÂBBP+dark) and photolyzed sample
(S166C-S-b 10ÂBBP+hv) were subjected to tryptic
digestion and peptide mapping, as described above for
the WT enzyme. For the photolyzed S166C-S-b
(10ÂBBP+hv) sample a reduction in intensity of the
tryptic fragment 6T (50%) was observed which was
accompanied by two new peaks, a minor peak desig-
nated as 6T0 and a major peak designated as 6T00. Both
6T0 and 6T00 were more hydrophobic than 6T itself and
showed mass increases over 6T of 197 and 188 Da
respectively. The tryptic fragments 6T0 and 6T00 were
isolated and subjected to amino acid sequencing. The
sequences for 6T0 and 6T00 were identical. The observed
Preparation of p-boronic acid benzophenone (1). A
stirred solution of 4-bromobenzophenone (3.12 g,
12.0 mmol), ethylene glycol (4.7 g, 75 mmol), and p-tol-
uene sulfonic acid (0.2 g, 1.2 mmol) in benzene (30 mL)
was heated under re¯ux for 48 h and the resulting water
was removed by azeotropic distillation in a Dean±Stark
trap.36,37 The reaction mixture was allowed to cool to
22 ꢀC and then aqueous 1 M NaOH (40 mL) was added
and the mixture was extracted with Et2O (3Â30 mL).
The organic phases were combined, dried (MgSO4), ®l-
tered, and then evaporated in vacuo yielding 4-bromo-
benzophenone ethylene glycol (2) as a white solid which
was recrystallized from EtOH (3.7 g, 12 mmol, quanti-
tative yield).36 1H NMR (200 MHz, CDCl3) d 4.03 (4H,
s, CH2), d 7.27±7.50 (9H, m, Ar-H) ppm.
. . .VANLS126 127xxx (BPN0 numbering) sequence was
L
identical to that derived from the 6T00 tryptic fragment
of the WT-SBL+BBP photolyzed sample. The observed
mass increase of 188 Da (BBP-2H2O:190 Da calcd) for
the tryptic fragment, is consistent with the formation of
a crosslink between the S166C-S-b and BBP with con-
comitant loss of two water molecules. However, it must
be noted that the mass of the intact S166C-S-b±BBP
complex does not demonstrate the concomitant loss
of two molecules of water. This is attributable to the
poorly resolved mass spectrum in this case (Table 3). The
identity of the minor tryptic fragment 6T0 is not appar-
ent. Both the WT and S166C-S-CH2-c-C6H11 enzymes
show a crosslink to BBP at residue Gly127 despite
having very di€erent active site environments. This
suggests that BBP forms a boronate ester adduct with
the enzyme, which although reversible is suciently
long-lived that it acts as a tether restricting the spatial
span of the BBP reagent in the enzyme active site.
Such adducts between boronic acid inhibitors and the
Ser221 Og hydroxyl of serine proteases have been
observed previously by X-ray crystallography33 and by
NMR.41
To a stirred solution of 4-bromobenzophenone ethylene
glycol (3), (1.0 g, 3.28 mmol) dissolved in dry THF
(10 mL) under N2 at 78 ꢀC was added drop wise via
syringe n-butyl lithium (1.7 mL of a 2.5 M solution in
hexane, 4.3 mmol).37 This mixture was stirred for 1 h
then transferred by cannula to a solution of tri-
methylborate (0.51 g, 4.9 mmol) in dry THF (10 mL) at
78 ꢀC.31,37 This mixture was stirred at 78 ꢀC for 1 h
and then allowed to warm to room temperature over-
night. The reaction mixture was slowly added to aqu-
eous 10% H2SO4 (20 mL) at 0 ꢀC and stirred for
30 min.31 The reaction mixture was then extracted with
Et2O (3Â30 mL). The combined organic phases were
dried (MgSO4), ®ltered and then evaporated in vacuo to
yield the boronic acid as a yellow oil which was added
directly to a ¯ask containing a solution of ethylene gly-
col (0.2 g, 3.3 mmol) in Et2O (25 mL) and stirred vigor-
ously for 1 h at 20 ꢀC.31 To this reaction mixture were
added hexanes (50 mL) and water (1 mL) and the aqu-
eous layer was separated. The organic layer was dried
(MgSO4), ®ltered, and then evaporated in vacuo yield-
ing the diglycal (4) as a white solid which was recrys-
Since no crosslinks were observed for S166C-S-a, -c,
-d after photolysis for 12 h in the presence of 10-fold
molar excess of BBP, each of these CMMs were photo-
lyzed for up to 72 h in the presence of a 80-fold molar
excess of BBP (the solubility limit). However, although
activity decreases were e€ected under these conditions,
puri®cation of the enzymes followed by their ESMS
analysis did not provide evidence of crosslinking. Thus
it was concluded that the introduced side chains of
S166C-S-a, -c and -d CMMs preclude proper orien-
tation or alignment of BBP in the S1 pocket for cross-
linking with the enzyme. The signi®cantly reduced
binding of BBP to S166C-S-a and -c, and the slightly
reduced binding of BBP to S166C-S-b and -d, as mani-
fested by higher KI values, and the lack of an observed
crosslink between BBP and S166C-S-a, -c and -d and
the longer reaction time, compared to WT, required
to e€ect a crosslink between S166C-S-b and BBP,
indicate that modi®cation of residue C166 makes the
S1 pocket less conducive to binding large P1 groups. It
is interesting that despite the fact that BBP binding
to S166C-S-b is 2-fold better than to S166C-S-d, the
former does not form a BBP crosslink while the
latter does. The reasons for this e€ect are unknown and
intriguing.
1
tallized from benzene (0.87 g, 2.9 mmol, 88% yield). H
NMR (200 MHz, CDCl3) d 4.06 (4H, s, CH2), 4.36 (4H,
s, CH2), 7.27±7.50 (9H, m Ar-H) ppm.
The diglycal 4, (270 mg, 0.91 mmol) was dissolved in a
stirred solution of 6 M HCl (15 mL) and acetone (5 mL)
and heated to 60 ꢀC for 2 h with stirring and then stirred
for a further 48 h at room temperature.37 The reaction
mixture was extracted with Et2O (2Â20 mL). The com-
bined organic phases were then washed with brine
(20 mL), poured into H2O (5 mL) and concentrated in
vacuo, with the benzophenone boronic acid precipitat-
1
ing as white crystals (180 mg, 0.8 mmol, 87% yield). H
NMR (200 MHz, DMSO-d6) d 8.35 (2H, s, OH), d 7.58±
7.98 (9H, m, Ar-H) ppm. 13C NMR (400 MHz, DMSO-
d6) d 196.2, 138.2, 137.1, 134.1, 132.8, 129.7, 128.6,
128.5 ppm. IR (KBr) 3500±3200 (B(OH)2), 2924 (aro-
matic CH) 1654 (carbonyl), 1318, 1284, 1113, 875,
704 cm 1, mp 103 ꢀC. HRMS for trimeric anhydride
C39H27BO3 calcd: 624.2086. Found: 624.2095.
Site-speci®c chemical modi®cation. To 25 mg of the
S166C mutant, puri®ed as previously described9,28 and

568
G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
stored ¯ash frozen in CHES bu€er (2.5 mL; 70 mM
CHES, 5 mM MES, 2 mM CaCl2, pH 9.5), at 20 ꢀC was
added in turn one of the methanethiosulfonate reagents
(2a±d) (100 mL of a 0.2 M solution) in a PEG (10,000)
coated polypropylene test tube, and the mixture
agitated in an end-over-end rotator. Blank reactions
containing 100 mL of solvent instead of the reagent
solution were run in parallel. Each of the modi®ca-
Research 96-09). The active enzyme concentration
determined in this way was used to calculate kinetic
parameters for each CMM.
Photolysis. Enzyme solutions for photolysis were
prepared in 3.5 mL quartz cuvettes and contained 3 mL
of enzyme dissolved (2 mg/mL) in storage bu€er
(20 mM MES, 1 mM CaCl2, pH 5.8). To each cuvette
was added 10 mL of p-boronic acid benzophenone (for
10Âsamples, 0.02 M BBP in DMSO). For the blank
reactions 10 mL of DMSO was added instead of BBP.
The solutions were photolyzed at 4 ꢀC in a Rayonet
photoreactor equipped with 350 nm lamps. The dark
controls were treated in the same way except the
cuvettes were wrapped in aluminum foil. At the time
intervals indicated, 10 mL aliquots were withdrawn,
diluted into 190 mL of storage bu€er and speci®c activity
measurements made. After photolysis, the enzymes were
puri®ed on a desalting column (Pharmacia Biotech
PD-10, Sephadex G-25 M) pre-equilibrated with MES
bu€er (3.5 mL, 5 mM MES, 2 mM CaCl2, pH 6.5). The
enzyme was eluted with MES bu€er, dialyzed against
storage bu€er (20 mM MES, 1 mM CaCl2, pH 5.8,
(3Â1 L)) at 4 ꢀC aliquoted, ¯ash frozen and stored at
20 ꢀC.
tion reactions was monitored spectrophotometrically
1 42
(E410=8800 M 1 cm
)
on a Perkin±Elmer Lambda 2
spectrophotometer, by speci®c activity measurements.
After the reaction was quenched by dilution with MES
bu€er (5 mM MES, 2 mM CaCl2, pH 6.5) at 0 ꢀC, the
speci®c activity of the CMM (10 mL) was determined in
bu€er (0.1 M Tris pH 8.6, 0.005% Tween 80 and 1%
DMSO, with the suc-AAPF-pNA as substrate (1 mg/mL,
purchased from Bachem Bioscience Inc.) at 25ꢀC. The
reaction was terminated when the addition of a further
100 mL of methanethiosulfonate solution e€ected no fur-
ther change in speci®c activity, generally within 30min.
The reaction solution was puri®ed on a disposable desalt-
ing column (Pharmacia Biotech PD-10, Sephadex G-25
M) pre-equilibrated with MES bu€er (5 mM MES,
2 mM CaCl2, pH 6.5) then dialyzed against 20 mM
MES, 1 mM CaCl2, pH 5.8 (3Â1 L) at 4 ꢀC and ali-
quoted into 0.5±1.5 mL volumes, ¯ash frozen in liquid
nitrogen and then stored at 20 ꢀC. Modi®ed enzymes
were analyzed by nondenaturing gradient (8±25%) gels
at pH 4.2, run towards the cathode on the Pharmacia
Phast-System according to Pharmacia Method 300, and
appeared as one single band.
Speci®c activity measurements for time course monitoring.
For speci®c activity determinations, absorbance versus
time measurements were made on a Perkin±Elmer
Lambda 2 spectrophotometer. The spectrophotometer
was zero'd against a 1 mL disposable cuvette containing
980 mL of pH 8.6 0.1 M Tris bu€er containing 0.005%
Tween 80 and 10 mL of the suc-AAPF-pNA substrate
(100.0 mg/mL in DMSO, 160 mM). The enzyme aliquot
(10 mL) was added and, after a 10 s delay, the absor-
Electrospray mass spectrometry. The CMMs were pur-
i®ed for ESMS analysis, by FPLC (BioRad, Biologic
System) on a Source 15 RPC matrix (17-0727-20 from
Pharmacia) with 5% acetonitrile, 0.01% TFA as the
running bu€er and eluted with 80% acetonitrile, 0.01%
TFA in a one step gradient. Electrospray mass spectra
were recorded on a PE SCIEX API III Biomolecular
Mass Analyzer. Mass: WT. Calcd: 26698. Found: 26694.
S166C-S-a: calcd: 26836. Found: 26832. S166C-S-b:
calcd: 26842. Found: 26844. S166C-S-c: calcd: 26853.
Found: 26851. S166C-S-d: calcd: 26714. Found: 26708
bance reading was started and sampled with an interval
1
of 0.2 s during 60 s at l=410 nM. The slope (in A s
)
was determined by linear curve ®t of the data below 5%
of substrate conversion. The rate of product formation,
dP/dt was determined using Beer's Law (A=Ecl, where
E
410=8800 M 1 cm 1, l=1 cm). The enzyme activity
1
was then calculated: speci®c activity (mmol min 1 mg
)
={(dP/dt M s 1)(60 s min 1)(106 mmol mol 1) (volume
in cuvette L)}/{[E] mg mL 1}.
Regeneration of unmodi®ed enzyme by treatment with ꢀ-
mercaptoethanol. To a solution of CMM (2.0 mg) in
250 mL of CHES-bu€er (70 mM CHES, 5 mM MES,
2 mM CaCl2, pH 9.5) was added 10 mL of a solution of
b-mercaptoethanol (1 M in 95% EtOH ). The reaction
was monitored by speci®c activity measurements and in
all cases the activity of the S166C cysteine parent was
restored.
Tryptic digests and HPLC-MS analysis.40 From a stock
solution of the SBL samples in storage bu€er (20 mM
MES, 1 mM CaCl2, pH 5.8) was withdrawn 0.1 mg
(ꢁ100 mL) of enzyme which was mixed with water
(ꢂ200 mL) and 1 N HCl (30 mL) to give a ®nal con-
centration of 0.03 mg/mL protein in 0.09 M HCl. This
mixture was chilled for 10 min on ice and any debris was
removed by centrifugation for 2 min at 14,000 rpm. The
supernatant was transferred to a clean Eppendorf tube
to which was added 35 mL TCA (50% w/v) to give a
®nal concentration of 5% (w/v) TCA. This mixture was
chilled for 15 min on ice and then the protein was col-
lected by centrifugation (2 min at 14,000 rpm). The pel-
let was washed once with 1 mL of 90% (v/v) acetone
( 20 ꢀC) and brie¯y dried in vacuo. The pellet was
resuspended in 12 mL of 0.5 M ammonium bicarbonate,
containing 8 M urea and then incubated at 37 ꢀC for
4 min. To this mixture was added 48 mL of 1% (w/v
Free thiol titration. The residual free thiol content of the
S166C CMMs, was determined spectrophotometrically
1
by titration with Ellman's reagent (E412=13,600 M
1 35
cm
)
in sodium phosphate bu€er (0.25 M, pH 8.0).
Active site titration. The active enzyme concentration
was determined as previously described43 by monitoring
¯uoride release upon enzyme reaction with a-toluene-
sulfonyl ¯uoride (Aldrich Chemical Co. Inc.) as mea-
sured by a ¯uoride ion sensitive electrode (Orion

G. DeSantis et al. / Bioorg. Med. Chem. 8 (2000) 563±570
569
in H2O) 1-O-octyl-b-d-glucopyranoside and 1 mL of
References
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adding 5 mL of TFA (10% v/v).
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1 42
(E410=8800 M 1 cm
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6
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We gratefully acknowledge ®nancial support from the
Natural Sciences and Engineering Research Council of
Canada (NSERC) and Genencor International Inc., and
the award of an NSERC scholarship (to G.D.). We are
indebted to Genencor International Inc. for supplying
us with the WT and S166C mutant of SBL. We are
grateful to Dr. Erika Plettner for technical assistance,
Sue Middlebrook of Genencor for peptide sequencing,
and Professors T. Tidwell and R. McClelland for the
use of their photoreactors.

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