(Received for publication, July 29, 1996, and in revised form, October 3, 1996)
From the Suntory Research Center, 1-1-1, Wakayamadai,
Shimamoto-cho, Mishima-gun, Osaka 618, Japan, the
§ Department of Nutritional Physiology, Faculty of
Nutrition, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku,
Kobe, 651-21 Japan, and the
Biomolecular Engineering Research
Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan
The Bacillus sp. SAM1606
-glucosidase with a broad substrate specificity is the only known
-glucosidase that can hydrolyze
,
-trehalose efficiently. The
enzyme exhibits a very high sequence similarity to the
oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose.
These three enzymes share 80% identical residues within the conserved
regions (CR), which have been suggested to be located near or at the
active site of the
-amylase family enzymes. To identify by
site-specific mutagenesis the critical residues that determine the
broad substrate specificity of the SAM1606 enzyme we compared the CR
sequences of these three glucosidases and selected five targets to be
mutagenized in SAM1606
-glucosidase, Met76,
Arg81, Ala116, Gly273, and
Thr342. These residues have been specifically replaced by
in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn,
respectively, as in the Bacillus O16G. The 12 mutant
enzymes with single and multiple substitutions were expressed and
characterized kinetically. The results showed that the 5-fold mutation
virtually abolished the affinity of the enzyme for
,
-trehalose,
whereas the specificity constant for the hydrolysis of isomaltose, a
good substrate for both the SAM1606 enzyme and O16G, remained
essentially unchanged upon the mutation. This loss in affinity for
trehalose was critically governed by a Gly273
Pro
substitution, whose effect was specifically enhanced by the
Thr342
Asn substitution in the 5-fold and quadruple
mutants. These results provide evidence for the differential roles of
the amino acid residues in the CR in determining the substrate
specificity of the
-glucosidase.
It has been shown that -amylases,
-glucosidases,
glucoamylases, cyclodextrin glucanotransferases, and pullulanases share several short conserved sequences (conserved regions,
CR1) (1-5) and have also been suggested to
have a common (
/
)8-barrel fold (6, 7). These enzymes
are also proposed to share a common reaction mechanism (3, 8, 9). These
characteristics suggest a strong evolutionary relationship in the
origin of these enzymes, which have thus been categorized into a single
protein family called the
-amylase family. X-ray crystallographic
studies of several members of this family showed that the CR are
located at or near the active site and contain putative catalytic
carboxylates (5, 10-12). Recent structural elucidation of
-amylases
and cyclodextrin glucanotransferase complexed with their substrates or
inhibitors has shown that some amino acid residues in these regions
indeed interact with the bound ligands (13-16). These observations indicate the significance of the CR sequences in maintaining catalytic activity and specificity of the enzyme.
-Glucosidase (EC 3.2.1.20) catalyzes the hydrolysis of
1-O-
-D-glucopyranosides with a net retention
of anomeric configuration. The substrate specificity of
-glucosidase
differs greatly with the source of the enzyme (17). The majority of
-glucosidases preferentially hydrolyzes maltose, whereas another
class of
-glucosidases, dextrin 6-
-glucanohydrolase
(oligo-1,6-glucosidase, O16G; EC 3.2.1.10), acts exclusively on the
-1,6-glucosidic linkage of isomaltooligosaccharides (18). We have
recently found that a strain of thermophilic Bacillus,
SAM1606, produced a novel thermostable
-glucosidase with a broad
substrate specificity and high transglucosylation activity (19). The
enzyme can hydrolyze efficiently a variety of
1-O-
-D-glucopyranosides such as
,
-trehalose (trehalose), maltose, nigerose, isomaltose, sucrose,
turanose, maltotriose, maltotetraose, and isomaltotriose. Indeed, it
was the first such
-glucosidase that could hydrolyze trehalose
efficiently. We cloned the SAM1606
-glucosidase gene to determine
its primary structure and expressed it in Escherichia coli
(20). SAM1606
-glucosidase exhibited sequence similarities to the
enzymes of the
-amylase family and contained the CR (Fig.
1) as well as a suggested (
/
)8-barrel fold. Thus, the enzyme was also assigned as a member of the
-amylase family. Unexpectedly, we found that the enzyme exhibits extremely high
sequence similarities (62-65% identity along the entire polypeptide chain and 80% identity within the CR sequences) to the O16G of Bacillus cereus and Bacillus thermoglucosidasius
(21-23). These O16G themselves are 72% (along the entire polypeptide
chain) or 80% (along the CR sequences) identical to each other but,
very interestingly, are distinct from SAM1606
-glucosidase in
substrate specificity; the O16G cannot hydrolyze trehalose, maltose,
and sucrose (18, 24, 25). Thus, limited structural differences within
the CR have been suggested to govern the significant differences in
substrate specificity.
In this study we have analyzed the broad substrate specificity of
SAM1606 -glucosidase by comparative site-specific mutagenesis. By
comparing the CR sequences between the SAM1606 enzyme and the Bacillus O16G, we selected five targets to be mutagenized in
the CR of SAM1606
-glucosidase: Met76,
Arg81, Ala116, Gly273, and
Thr342. These residues have been specifically replaced by
Asn, Ser, Val, Pro, and Asn, respectively, by in vitro
mutagenesis, as are found in the O16G (Fig. 1). The mutant enzymes with
their single and multiple substitutions were overexpressed and
characterized kinetically. The results showed that the multiple
mutations containing Gly273
Pro and Thr342
Asn caused an alteration in substrate specificity: affinity for
trehalose has been specifically diminished upon the mutations. We
describe here the mutational analyses of SAM1606
-glucosidase to
identify Gly273
Pro as a critical substitution for
differential specificity between the SAM1606 enzyme and O16G and find
enhancement of its effect by Thr342
Asn.
Chemicals
p-Nitrophenyl -D-glucopyranoside,
maltose, isomaltose, sucrose, and trehalose, all of analytical grade,
were obtained from Nacalai Tesque, Kyoto, Japan. Maltose was freed from
contaminant glucose by high performance liquid chromatography on an
Asahipak NH2P50 column (1 × 25 cm) using a Shimadzu LC9A system
in which 70% (v/v) CH3CN in H2O was
isocratically developed at a flow rate of 2.0 ml/min by monitoring
using thin layer chromatography (19). To find the exact concentration
of isomaltose, which is supplied as a syrup containing water,
isomaltose was hydrolyzed in 1 M HCl at 100 °C for
24 h. After neutralization with NaOH, the glucose formed was
determined by the method of Pütter and Becker (26) using a kit
(Boehringer Mannheim). For all other chemicals, the purest reagents
available were used.
Bacterial Strains and Plasmids
Plasmid pGBSU2, a derivative of pGBSU1 (20), was used as a
template in in vitro mutagenesis of the -glucosidase
gene. E. coli strain W3110 was used for expression of the
wild type and all of the mutant
-glucosidases.
Site-directed Mutagenesis and Expression
A strategy for in vitro mutagenesis is depicted in
Fig. 2A. Fragments I, II, III, and IV of the
SAM1606 -glucosidase gene were amplified by PCR using a template
plasmid pGBSU2 (Fig. 2B), and the PCR primers (see also
Table I) which were so designed that the restriction
enzyme sites were newly created at boundaries of neighboring fragments
without a change in the amino acid sequences. Fragments I, II, III, and
IV were ligated with each other, and the resultant fragment was
substituted for the BamHI-AatII fragment of the
pGBSU2 to obtain pGBSU5 (Fig. 2C). Replacement of the
fragment(s) in the pGBSU5 with the mutated fragment(s), whose
preparations are described below, allowed us to obtain
-glucosidase
genes having various combinations of mutations. The mutations
Met76
Asn, Arg81
Ser,
Ala116
Val, and their double and triple mutations were
introduced on the amplified fragment II essentially as described by
Kramer and Frits (27) using the mutagenesis primers M1,
M2, and M3. For the mutation Gly273
Pro, fragments IIIa and IIIb were amplified from pGBSU2 using PCR
primers F-IIIa/R-IIIa and F-IIIb/R-IIIb, respectively, which were so
designed that the amplified fragments were to be ligated at a newly
created SmaI site, giving rise to the substitution of Pro
for Gly273. The mutation Thr342
Asn was
introduced by PCR, which was directly performed on the pGBSU2 with PCR
primers M5 (instead of F-IV) and R-IV. Individual mutations
were verified by DNA sequencing.
|
Mutant and wild type -glucosidase genes were expressed in E. coli W3110 transformant cells under the control of the
icp promoter of the insecticidal protein gene from B. thuringensis subsp. sotto as described (20, 28).
E. coli transformant cells were grown to the stationary
phase at 37 °C in 5 liters of L-broth containing 50 µg/ml
ampicillin, and the cells were collected by centrifugation.
Enzyme Purification
The wild type and all of the mutant -glucosidases were
purified as described below. All steps were done at 4 °C unless
otherwise stated. Sodium phosphate buffer (0.01 M, pH 7.0)
was used as the standard buffer. Enzyme activity was routinely found by
assay method I (see below). Cells (typically 20 g, wet weight)
were ground with 40 g of aluminum oxide powder for 10 min in a
mortar chilled on ice and suspended in 40 ml of standard buffer
followed by centrifugation. Polyethyleneimine was added to the
supernatant at a final concentration of 0.12% (w/v). After the mixture
was left for 30 min, the precipitate was removed by centrifugation. The
supernatant was then kept at 60 °C for 30 min. After the heat treatment, the precipitate was removed by centrifugation. To the supernatant solution, solid ammonium sulfate was added slowly to 20%
saturation. After the mixture was allowed to stand for 1 h, the
insoluble material was removed by centrifugation. Sodium ammonium
sulfate was then added to 50% saturation. The precipitate, 20-50%
saturated fraction, was dissolved in the standard buffer and dialyzed
against the standard buffer. The insoluble material formed during the
dialysis was removed by centrifugation. The enzyme solution was loaded
on a DEAE-Sepharose CL-6B column (3.6 × 21 cm) equilibrated with
the standard buffer. The enzyme was eluted with a linear gradient of
NaCl (0-0.6 M) in the same buffer (400 ml each). Active
fractions were analyzed for the subunit structure of the enzyme by
native- and SDS-polyacrylamide gel electrophoresis (PAGE). It was found
that at this step, a small fraction of the monomeric form of the enzyme
could be separated from multimeric forms. Fractions containing >90%
pure monomer were collected and concentrated with PM10 membrane in an
Amicon 8200 ultrafiltration unit. The concentrate was put on a
Sephacryl S200 HR column (2.6 × 67 cm) equilibrated with the
standard buffer containing 0.1 M NaCl and eluted. The
monomeric
-glucosidase fractions were combined, concentrated by
ultrafiltration, dialyzed against the standard buffer, and used for
kinetic analyses.
Enzyme Assay
Method IThe enzymatic hydrolysis of
p-nitrophenyl -D-glucopyranoside was
monitored by the amount of p-nitrophenolate released at 55 °C. The standard assay mixture contained 1.0 µmol of
p-nitrophenyl
-D-glucopyranoside, 10 µmol
of sodium phosphate buffer, pH 7.2, and the enzyme in a final volume of
1.0 ml. The mixture without the enzyme was brought to 55 °C. The
reaction was started by the addition of enzyme, and changes in
absorbance at 405 nm were recorded with a spectrophotometer (Shimadzu
UV-160; kinetic mode) equipped with a temperature-controlled cell
positioner (CPS240A). The extinction coefficient for
p-nitrophenolate under these conditions was 13,400 cm
1 M
1 (19).
For the kinetic analysis for hydrolysis of
trehalose, maltose, sucrose, and isomaltose, the reaction mixture
contained varying amounts of sugar, 1.0 µmol of sodium phosphate
buffer, pH 7.2, and the enzyme in a final volume of 100 µl. The
mixture without the enzyme was brought to 55 °C. The reaction was
started by the addition of enzyme. After incubation at 55 °C for 10 min, the reaction was stopped by heating at 100 °C for 3 min.
Glucose that formed in the reaction mixture was determined by the
method of Pütter and Becker (26) with a kit (Boehringer
Mannheim). The blank did not contain the enzyme. One unit of the enzyme
is defined as the amount of enzyme which catalyzes the hydrolysis of 1 µmol of substrate/min at 55 °C. Km and
Vmax values and their standard errors were
estimated by fitting the initial velocity data to the Michaelis-Menten
equation by nonlinear regression methods (29). The absorption
coefficient of the purified SAM1606 -glucosidase,
A280[1%] = 25.5, which
was calculated from the amino acid sequence (20), was used for unit
calculations.
pH Activity Profiles
Enzymatic hydrolyses of trehalose, maltose, sucrose, and isomaltose were assayed by method II except that the reaction mixtures contained 5.0 µmol of substrate and 1.0 µmol of either sodium acetate, pH 2.0-6.0, or sodium phosphate, pH 6.0-8.0.
Analytical Methods
Native-PAGE and SDS-PAGE were done with a 10% gel by the
procedures of Davis (30) and Laemmli (31), respectively. Proteins were
stained with Coomassie Brilliant Blue R-250 and destained in a
destaining solution (a 2:1:7 mixture of methanol, acetic acid, and
water). For Western blotting, proteins in the SDS-PAGE gel were
transferred to a nitrocellulose membrane, which was then blocked with
1% bovine serum albumin in phosphate-buffered saline, pH 7.2, at room
temperature for 2 h. The blots were probed with a 1:400 dilution
of the primary anti-SAM1606 -glucosidase antiserum (rabbit) and the
recommended dilution of secondary horseradish peroxidase coupled to
goat anti-rabbit immunoglobulin G (Bio-Rad). The immune complexes were
visualized by the peroxidase-catalyzed oxidation of 4-chloro-1-naphthol
by hydrogen peroxide. The molecular weights of mutant and wild type
-glucosidases were estimated by gel permeation chromatography on TSK
gel G3000SW-XL (0.7 × 25 cm) equilibrated with 0.01 M
sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl.
The amino acid sequence from the amino terminus was determined by
automated Edman degradation with a Shimadzu gas-phase sequencer (model
PPSQ-10).
A series of mutant enzymes of SAM1606 -glucosidase was
constructed to probe amino acid residues as determinants for the
specificity of the SAM1606 enzyme. The sites of mutagenesis and
replacing amino acids were selected by comparing the CR sequences
between the SAM1606 enzyme and O16G which exhibit striking sequence
similarities but have distinct differences in their substrate
specificity. We established a convenient system for construction and
expression of mutant enzymes with all of the possible combinations of
mutations (Fig. 2). Twelve of these mutants (Table II)
were generated, all of which were expressed in E. coli cells
and existed as both the monomeric and multimeric forms as the wild type
enzyme. All of the mutant enzymes are stable during heat treatment at
60 °C and pH 7.0 for 30 min as is the wild type enzyme, and this
permitted their efficient purifications. At the ion exchange
chromatography step, a small fraction of the monomeric form could be
separated from multimeric forms. The monomeric form thus separated was
purified further to homogeneity by gel filtration chromatography on the criterion of native-PAGE. All of the purified mutant enzymes were eluted as a single peak corresponding to the expected native molecular weight (Mr 68,000), as confirmed by analytical
gel permeation chromatography on TSK gel G3000SW, and were
cross-reacted with polyclonal antibody raised against the wild type
enzyme, as determined by Western blotting analysis. Amino-terminal
amino acid sequence analyses up to 10 cycles of the wild type and all
of the mutant enzymes confirmed the expected primary structure
Ser-Thr-Ala-Leu-Thr-Gln-Thr-Ser-Thr-Asn (20).
|
The steady-state kinetic parameters for hydrolysis of four different
substrates, trehalose, maltose, sucrose, and isomaltose, were
determined for the wild type and mutant enzymes at pH 6.0 and are given
in Table III. Among these substrates, trehalose is known
to be a very poor substrate for most known -glucosidases including
the Bacillus O16G but has been shown to be hydrolyzed effectively by SAM1606
-glucosidase. Maltose and sucrose were also
substrates for which distinct differences in reactivity have been
established between the SAM1606 enzyme and O16G, whereas isomaltose can
serve as an excellent substrate for both enzymes.
|
None of the single and multiple mutations caused a significant reduction in Vmax for all of the substrates tested; all mutant enzymes had Vmax values that were more than 20% of those of the wild type enzyme for all substrates. Some mutant enzymes exhibited even higher specific activities than that of the wild type enzyme. These are consistent with the fact that the mutagenic targets selected in this study did not contain the putative catalytic residues.
No significant variation in Km was detected with
maltose, sucrose, and isomaltose upon each mutation. For trehalose, however, Gly273 Pro as well as all multiple mutations
containing the Gly273
Pro mutation had distinct effects
on Km from those obtained by the other mutations;
only these mutations caused appreciable increases in the
Km value for this substrate. In contrast, a
quadruple mutant without the Gly273
Pro substitution
(i.e. Q4
) showed a Km value for trehalose which was similar to that of the wild type enzyme. These results indicate that the increase in Km for
trehalose is critically governed by the Gly273
Pro
substitution, which solely caused a 10-fold greater increase in the
Km value than those of mutants without it, as shown
by comparison of Km values for trehalose of
S4 and the other single mutants. In addition, the results
with mutants with four and five alterations also indicate that the
effect of the Gly273
Pro substitution was enhanced
further by a Thr342
Asn substitution in these mutants;
the presence of Asn342 in these cases (i.e. Q1
,
Q2
, Q3
, and F) caused more than
10-fold additional increases in Km for trehalose
than with the Gly273
Pro mutants without
Thr342
Asn substitution (i.e. S4 and
Q5
, Table III). Interestingly, however, such an enhancement
was not observed in the D4/5 mutant where the
Thr342
Asn substitution was only introduced into the
single Gly273
Pro mutant (S4). It should be
emphasized that, judging from the Km values,
Q1
, Q2
, Q3
, and F cannot
bind trehalose under the assay conditions that have been employed
routinely with a relatively low substrate concentration
(i.e. 5 mM; Ref. 19), albeit their
Km values for isomaltose were almost unchanged (Table III).
To find the net changes in substrate preference of the enzyme upon
mutations, we compared the relative specificity constants using
isomaltose as the reference substrate (Fig. 3);
isomaltose showed the least variation in the specificity constant upon
all mutations (Table III), consistent with the fact that this sugar serves as a good substrate for both the SAM1606 enzyme and O16G. The
largest changes in the substrate specificity were detected with mutant
enzymes exhibiting exclusive diminutions in the relative specificity
constant for trehalose.
We also examined the apparent pH activity profiles for hydrolysis of these substrates with six representative mutants (S1, S2, S3, S4, S5, and F) to address the possibility that the observed change in the substrate specificity is due to a change in the pH dependence of the hydrolysis reaction upon mutations. Optimum pH values for hydrolysis of trehalose, maltose, and sucrose were at 5.5 for all six mutant enzymes and were unchanged from those of the wild type enzyme, although some variations in the optimum pH were observed with isomaltose. Optimum pH values were at 4.7 for the wild type enzyme, S1, S2, and S5, and at 3.5 for F; but S3 and S4 showed a broad optimum pH ranging from 4.5 to 6.0. Thus, the change in the specificity was not due to a specific shift in the pH optimum for trehalose hydrolysis.
The strategy we have taken in this study to probe amino acid
residues responsible for the uniquely broad substrate specificity of
the SAM1606 -glucosidase can be called comparative site-specific mutagenesis; the sites and amino acids chosen for replacement were
selected by comparing the CR sequences with those of reference enzymes
that show high sequence similarities but have distinct and narrower
substrate specificities. This strategy is based on the recent reports
that the CR sequences are at or near active and substrate binding sites
of the enzyme and are suggested to be important in determining the
specificity of the enzyme (5, 10-16). The O16G of B. thermoglucosidasius and B. cereus were very good
reference enzymes for the SAM1606 enzyme for this purpose because they
are 80% identical to the SAM1606 enzyme in the CR but are very
different in terms of substrate specificity from the SAM1606 enzyme
(19, 20). Five nonconserved amino acids, Met76,
Arg81, Ala116, Gly273, and
Thr342, were identified and selected for mutagenesis and
were replaced with Asn, Ser, Val, Pro, and Asn, respectively. Enzymes
with all possible combinations of one through five mutations could be
constructed easily and expressed in our established system.
In this study, examination of 12 of the possible mutant enzymes
successfully led us to find that replacing Gly273 with Pro
caused a significant and specific diminution of the affinity of an
enzyme for trehalose without a significant decrease in
Vmax value and thus permitted us to identify
Gly273 as a critical determinant for differential
reactivity to trehalose between the SAM1606 enzyme and O16G. The
present studies also established the role of other amino acid residues
in the CR in determining the specificity of the enzyme; the
Thr342 Asn substitution in the mutants with four and
five alterations is important in enhancing the effect of the
Gly273
Pro substitution. Thus, the specificity of the
-glucosidase for trehalose arises from two distinct types of effects
of amino acid residues. One of them determines critically the
specificity, and the other enhances the effect of the
Gly273 without any critical effect by itself. The latter
effect by the Thr342
Asn substitution was observed in
the four- and five-substitution mutants, but not in the double mutant
(D4/5), suggesting that the enhancement effect of
the Thr342
Asn substitution emerged in the enzymes with
the CR sequences that are more similar to those of O16G than to that of
the SAM1606 enzyme. Although the SAM1606 enzyme is the only known
-glucosidase that can efficiently act on trehalose (19, 20), these
results suggest that this uniqueness is simply ascribed to the
exclusive ability of the SAM1606 enzyme to bind this sugar efficiently. This implies that O16G and probably the other
-glucosidases of the
same family lack the ability to bind trehalose because of their
different CR sequences; however, they might be engineered genetically
to hydrolyze trehalose by enabling them to bind trehalose through
appropriate substitution of amino acid residues in their CR sequences,
because these
-glucosidases are proposed to share a common reaction
mechanism for cleaving the
-glucosidic linkages (3, 8, 9) and thus
potentially allow hydrolysis of the
-1,1-linkage.
It should be pointed out that the effects of mutations on kinetic
parameters varied with the substrates; contrary to the results obtained
with trehalose, less variation in kinetic parameters was observed with
maltose, sucrose, and isomaltose by mutations introduced in this study.
Although distinct differences in reactivity have been established for
maltose and sucrose between the SAM1606 and O16G enzymes, these
differences could not be explained fully in terms of amino acid
substitutions within the CR sequences. The current results indicate
that replacement of the amino acid residues within the CR causes
distinct effects on the reactivity of each substrate and suggest that
critical amino acid residue(s) determining the reactivity to individual
substrates may vary with the substrate. These conclusions are
consistent with observations from x-ray crystallographic studies of
several -amylase family enzymes complexed with substrates and
inhibitors (13-15). (i) Binding of a ligand to the enzyme is
maintained through many polar and nonpolar protein-ligand interactions,
including a hydrogen bonding network, which is in many cases engaged in
interactions with the solvent water. (ii) Binding of a different ligand
produces a different set of interactions. Thus, the five-substitution
mutation may disrupt the interactions necessary for trehalose binding
but may not essentially affect those interactions necessary for binding and subsequent catalytic steps in the hydrolysis of maltose, sucrose, and isomaltose, although it does somewhat perturb the pH dependence of
isomaltose hydrolysis. Reactivity to sucrose and maltose of the SAM1606
enzyme may be governed by amino acid residue(s) other than the sites
selected for mutagenesis in this study. Knowledge of the interactions
between substrate and enzyme in the stereostructure of the SAM1606
-glucosidase-substrate complex will be necessary to elucidate
further the broad substrate specificity of this enzyme.
Gly273 and Thr342 are located near the putative
catalytic residues of SAM1606 -glucosidase, Glu271 and
Asp345, and are positioned at less conserved sites in the
CR of
-amylase family enzymes, implying their potential roles in
defining the specificity. It is very interesting to find amino acid
residues at positions in the CR corresponding to those of the
Gly273 and Thr342 of the SAM1606 enzyme in the
other enzymes of
-amylase family and their interactions with bound
ligand in the reported stereostructures of the enzyme-inhibitor
complexes. This is exemplified by the recent x-ray crystallographic
studies of the porcine pancreatic
-amylase complexed with acarbose,
a pseudosaccharidal inhibitor (13), and that with Temdamistat, a
proteineous inhibitor (14). In this
-amylase, Ile235 and
Asp297, respectively, correspond to the Gly273
and Thr342 of the SAM1606 enzyme (Fig. 1) and are located
near the bound inhibitors. Particularly, Ile235 in the
porcine pancreatic
-amylase appears to be in a close, though
indirect, contact with the bound inhibitors. These observations corroborate the importance of Gly273 in substrate binding
and the remarkable effect of its replacement with Pro.