(Received for publication, November 24, 1994; and in revised form, January 19, 1995)
From the
Determination of the crystal structures of a
1,3--D-glucanase (E.C. 3.2.1.39) and a
1,3-1,4-
-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J. N, Garrett, T. P. J., Colman,
P. M., Chen, L., Høj, P. B., and Fincher, G. B.(1994) Proc.
Natl. Acad. Sci. U. S. A. 91, 2785-2789) showed the spatial
positions of the catalytic residues in the substrate-binding clefts of
the enzymes and also identified highly conserved neighboring amino acid
residues. Site-directed mutagenesis of the 1,3-
-glucanase has now
been used to investigate the importance of these residues. Substitution
of glutamine for the catalytic nucleophile Glu
(mutant
E231Q) reduced the specific activity about 20,000-fold. In contrast,
substitution of glutamine for the catalytic acid Glu
(mutant E288Q) had less severe consequences, reducing k
approximately 350-fold with little effect on K
. Substitution of two neighboring and
strictly conserved active site-located residues Glu
(mutant E279Q) and Lys
(mutant K282M) led to 240-
and 2500-fold reductions of k
, respectively,
with small increases in K
. Thus, a tetrad
of ionizable amino acids is required for efficient catalysis in barley
-glucanases. The active site-directed inhibitor 2,3-epoxypropyl
-laminaribioside was soaked into native crystals. Crystallographic
refinement revealed all four residues (Glu
,
Glu
, Lys
, and Glu
) to be in
contact with the bound inhibitor, and the orientation of bound
substrate in the active site of the glucanase was deduced.
The enzymic hydrolysis of polysaccharides and glycosides is a critically important process in the metabolism of plants, animals, and microorganisms (Sinnott, 1990). The basic mechanistic assumption, supported with few exceptions by the available data, is that enzyme-mediated glycoside hydrolysis is initiated by protonation of glycosidic oxygen atoms via an active site-located general acid. Following protonation, the aglycon is released, and a positively charged oxycarbonium ion is stabilized by a nucleophilic amino acid, most often a carboxylate (reviewed by Svensson and Søgård(1993)). The exact mode of stabilization is not known but is thought to be either electrostatic or via a covalent enzyme-substrate intermediate. Hydration of the stabilized oxycarbonium ion or, alternatively, hydrolysis of the covalent enzyme-substrate intermediate serves to regenerate the reducing terminus of the glycosyl moiety.
Amino acids mediating in the protonation and stabilization are referred to as the catalytic acid and the catalytic nucleophile, respectively. Identification of such catalytic residues has been attempted in a number of enzymes using chemical modification (Svensson et al., 1990), x-ray crystallography (Anderson et al., 1981), or homology searches coupled with site-directed mutagenesis of residues thought to be involved (Trimbur et al., 1992). Due to theoretical and practical constraints in each of these techniques, a combined approach is required for a reliable definition of the functional importance of individual amino acids at the active sites of enzymes (Schimmel, 1993).
To define the molecular basis for catalysis and substrate
specificity in polysaccharide endohydrolases, we have employed a range
of mechanism-based inhibitors to define geometric differences within
the active sites of a family of -glucan endohydrolases (Høj et al., 1989b, 1991, 1992). In particular, detailed
investigations of 1,3-
-D-glucan glucanohydrolases (E.C.
3.2.1.39) and 1,3;1,4-
-D-glucan 4-glucanohydrolases (EC
3.2.1.73), two classes of polysaccharide hydrolases with closely
related substrate specificities, led to the conclusion that the
acquisition of distinct substrate specificities in the evolution of
these related enzymes did not require the recruitment of novel
catalytic amino acids but rather that it involved differences in the
positioning of catalytic residues at the active site and/or changes in
residues responsible for substrate binding (Chen et al., 1993a).
A 1,3--glucanase, designated isoenzyme GII, (
)and a 1,3;1,4-
-glucanase, designated isoenzyme EII,
have recently been crystallized (Chen et al., 1993b).
Their three-dimensional structures reveal a complete spatial
conservation of both the catalytic nucleophile (Glu
in
the 1,3-
-glucanase and Glu
in the
1,3-1,4-
-glucanase) and the proposed general acid
(Glu
in both enzymes) (Varghese et al., 1994). To further investigate the structural basis for
catalysis and substrate specificity in these enzymes, we have now
established bacterial expression systems for both classes of enzyme and
used site-directed mutagenesis of the 1,3-
-glucanase to determine
the relative importance of these putative catalytic residues in enzyme
action. Inspection of the three-dimensional structures reveals that the
catalytic amino acids Glu
and Glu
are
spatially close to the functional groups of Glu
and
Lys
. We have also tested the contribution of these two
neighboring conserved residues (Glu
and
Lys
) and conclude they play an important role in binding
and/or catalysis. This conclusion was independently corroborated by
structural studies, which revealed that Glu
and
Lys
are in intimate contact with a mechanism-based
inhibitor covalently linked to the nucleophile Glu
.
Primer A is identical to
the nucleotides encoding Ile of the
1,3-
-glucanase, while primer B is complementary to the 3`-end of
the cDNA (nucleotides TTC
to GCT
) but is
extended to incorporate an EcoRI site in the PCR product. For
PCR, reaction mixtures containing 30 ng of template, 150 pmol of each
primer, 300 µM each of dNTPs, and 1 unit of Vent
DNA polymerase in 50 µl of Vent
buffer
(consisting of 10 mM KCl, 10 mM (NH
)
SO
, 20 mM Tris-HCl buffer (pH 8.0), 2 mM MgSO
, and 0.1%
Triton X-100) were heated for 5 min at 94 °C followed by 25 cycles
of denaturation (94 °C, 60 s), annealing (45 or 55 °C, 60 s),
and extension (72 °C, 90 s). Amplified PCR fragments were purified
by agarose gel electrophoresis, cut with restriction enzyme EcoRI, and ligated into the pMAL-c2 vector previously cut with XmnI and EcoRI to generate the 1,3-
-glucanase
isoenzyme GII expression vector pMAL-c2-GII, which was used to
transform E. coli strain DH5
(supE44
lacU169(
80lacZ
M15)).
Colonies were grown on an LB agar plate containing 100 µg/ml
ampicillin and restreaked onto a master LB ampicillin plate and an LB
ampicillin plate containing 0.02% X-gal and 100 µM IPTG
for selection of white colonies. This procedure was adopted because the
presence of 100 µM IPTG on the first selection plate
drastically reduced the number of transformants. Subsequent experiments
revealed that reducing the IPTG concentration to 10 µM diminished this problem. Recombinant clones expressing the MBP-GII
fusion protein were further selected by activity measurements and
electrophoretic analysis of unpurified bacterial extracts.
Expression vectors directing the synthesis of barley
1,3-1,4--glucanase isoenzymes EI and EII were constructed
exactly as described above, using cDNAs encoding the respective enzymes
(Fincher et al., 1986; Slakeski et al., 1990). The forward primer A for both cDNAs was
5`-ATCGGGGTGTGCTACGGCATGAGC-3`, and the reverse primers B for
isoenzymes EI and EII were 5`-CCGGGAATTCCGTAGCTGCTACCTCATCAGAA-3` and
5`-GGCGGAATTCCCGAGCACGAGCTCCGTCAGAA-3`, respectively. Strains
expressing MBP-GII, MBP-EI, and MBP-EII are referred to as
DH5
MBP-GII, DH5
MBP-EI, and DH5
MBP-EII, respectively.
For
purification of the wild-type enzyme, crude extract from 1 liter of
culture was diluted 10-fold with 15 mM Tris-HCl buffer, pH
8.0, and applied at a flow rate of 2.5 ml/min to a DEAE-Sepharose fast
flow (Pharmacia) column (3 11.5 cm) equilibrated with 25 mM Tris-HCl buffer, pH 8.0. After washing the column exhaustively,
bound proteins were eluted with a linear 0-250 mM NaCl
gradient in 1.2 liters of equilibration buffer. Fractions containing
enzyme activity were pooled, desalted, and adjusted to 25 mM sodium acetate buffer, pH 5.0, by ultrafiltration (Amicon PM10
filter) before application to a CM-Sepharose fast flow (Pharmacia)
column (2
10.5 cm) equilibrated with 25 mM sodium
acetate buffer, pH 5.0. After exhaustive washing, bound proteins were
eluted with a linear 0-200 mM NaCl gradient in 1 liter
of equilibration buffer. Fractions containing pure protein were pooled
to give 5.0 mg of active fusion protein.
Mutant enzymes were
purified by a single ion-exchange chromatography step employing a
shallow salt gradient elution. The crude extract from 4-5 liters
of culture was diluted 10-fold with 15 mM Tris-HCl buffer, pH
8.0, and applied at a flow rate of 2.5-3.0 ml/min to a
DEAE-Sepharose column (5 21 cm) equilibrated with 12.5 mM Tris-HCl buffer, pH 8.5. After exhaustive washing, bound proteins
were eluted with a 1.9-liter linear 0-80 mM NaCl
gradient at a flow rate of 2.0 ml/min. Fractions containing pure fusion
protein were located by SDS-polyacrylamide gel electrophoresis, pooled,
concentrated, and adjusted to 25 mM sodium acetate buffer, pH
5.0, by ultrafiltration before clarification by centrifugation. The
yield of pure fusion protein varied between 10 and 20 mg, depending on
the mutation introduced.
Thermostabilities of the purified enzymes were determined by measuring residual activity after incubation in 50 mM sodium acetate buffer, pH 5.0, containing 0.5 mg/ml bovine serum albumin at 50 or 55 °C. Analytical gel filtration chromatography was carried out at room temperature using a Superose-12 HR 10/30 fast protein liquid chromatography column (Pharmacia) operated at a flow rate of 0.5 ml/min in 35 mM sodium acetate buffer, pH 5.0, containing 0.1 M NaCl.
Figure 1:
Purification of
MBP-glucanase fusion proteins synthesized in E. coli.
SDS-polyacrylamide gel electrophoresis analysis was performed, and
proteins were visualized with Coomassie Brilliant Blue. Lane
1, molecular weight markers; lane 2, extract from
non-induced DH5MBP-EI; lane 3, extract from IPTG-induced
DH5
MBP-EI; lane 4, extract from IPTG-induced
DH5
MBP-EII; lane 5, extract from IPTG-induced
DH5
MBP-GII; lane 6, MBP-GII pool after DEAE-Sepharose
step; lane 7, MBP-GII pool after CM-Sepharose step, lane
8, extract from IPTG-induced DH5
MBP-GII(E279Q); lane
9, purified MBP-GII(E279Q).
Attempts to cleave the
purified fusion protein with factor Xa protease were unsuccessful, a
result which can now be explained by inspection of the
three-dimensional structures of the 1,3- and
1,3-1,4--glucanase. The amino-terminal residue of each
enzyme is almost totally buried within the core of the
/
barrel structures (Varghese et al., 1994) (Fig. 2).
Access of factor Xa to the scissile bond in the folded fusion proteins
is therefore not possible.
Figure 2: Schematic view of barley endoglucanases showing the orientation of the catalytic groove with respect to MBP. The main chain of isoenzyme GII showing secondary structure elements as determined (Varghese et al., 1994) is shaded. The closedarrow points to the amino terminus, the point of attachment of MBP, while the openarrow points to the extended substrate binding cleft and the catalytic site.
Figure 3:
pH
optima for wild-type and mutant enzymes. Isoenzyme GII purified from
germinating barley (), MBP-GII(wt) (
), MBP-GII(E288Q)
(
), MBP-GII(K282M) (
), and MBP-GII(E279Q) (
) were
incubated at the indicated pH values, and their relative activities
were measured. Background activities were obtained at all pH values
tested and subtracted.
Figure 4:
Thermostabilities of endoglucanases
synthesized in germinating barley or in E. coli as fusion
proteins. A, isoenzymes GII (0.7 µg/ml) () and EII
(18 µg/ml) (
) were purified from germinating barley and
incubated at 50 °C in 50 mM sodium acetate (pH 5.0) in the
presence of bovine serum albumin (0.5 mg/ml). Residual enzyme
activities (A
) were determined and
compared to the initial activity at t = 0 (A
). B, as in A, except
MBP-GII (3.1 µg/ml) (
) and MBP-EII (39 µg/ml) (
)
were used.
Figure 5:
Invariant active site residues in some
plant and fungal endoglucanases. A, relevant segments of the
deduced primary structures of barley 1,3--glucanases isoenzyme GI
to GVII (Xu et al., 1992; Malehorn et al., 1993), a
bean (Phaseolus vulgaris) 1,3-
-glucanase (Edington et
al., 1991), a tobacco (Nicotiana tabacum)
1,3-
-glucanase (Payne et al., 1990), barley
1,3-1,4-
-glucanases isoenzyme EI (Slakeski et al.,
1990) and EII (Fincher et al., 1986), and a Saccharomyces
cerevisiae (Sc) 1,3-
-glucanase (Klebl and
Tanner, 1989) were visually aligned, and residues with positional
identity in all of these enzymes are shaded. The position of
the catalytic nucleophile (Glu
(GII)) is indicated with
the arrow (&cjs0435;), and the catalytic acid is indicated
with a star (
). The mutated residues
(Glu
, Glu
, Lys
, and
Glu
) are indicated with
. B, deduced amino
acid sequence for a portion of the anther-specific protein encoded by
the B. napus A6 gene (Hird et al., 1993) with no
apparent glucanase activity. Residues with positional identity to the
invariable positions of the 30-35-kDa endoglucanases shown in panelA are shaded. The non-conserved
Gln
residue is indicated with
(see
text).
Figure 6:
Active site structure of isoenzyme GII and
location of a covalently linked substrate analogue. A, the
inhibitor is in thickbonds (carbon, gold;
oxygen, crimson), and the protein is shown as a smoothed
C trace (gray) with active site chains as balls and sticks (carbon, white; nitrogen, paleblue; oxygen, pink). The inhibitor is covalently
attached to the catalytic nucleophile (Glu
, in red (bottomcenter)) behind the inhibitor, and the
oxygen, originally from the epoxide ring, is hydrogen bonded to the
catalytic acid (Glu
in red) via a water
molecule. The glucose moiety modeled is hydrogen bonded via O-6 to
Glu
and Lys
and via O-2 to
Asn
. There is hydrophobic contact between the face of the
glucose and the ring of Tyr
. The catalytic acid
(Glu
) and nucleophile (Glu
) are shown in red. Side chains included are Cys
,
Gly
, Ile
(center to upperleft); Tyr
, Phe
(left); Gly
, Asn
(frontleft); Asn
, Glu
(frontright); Asn
(behind Glu
),
Tyr
, Phe
(right);
Glu
, Phe
, Glu
,
Lys
, Glu
, and Phe
. B, stereo view of panelA with the
non-reducing end of the glucosyl moiety facing
North.
However, as pointed
out by Schimmel(1993), what appear to be straightforward rationales for
the conservation of residues at a particular location may be secondary
to alternative and more sophisticated considerations and that no matter
how obvious the role of a particular residue may seem, it pays to make
substitutions at such positions to test the effect on function, as
illustrated by the analysis of Asn in thymilidate
synthase (Liu and Santi, 1993). For these reasons the putative
catalytic amino acid residues of the barley 1,3-
-glucanase were
subjected to site-directed mutagenesis, the enzymes were expressed in E. coli, and the kinetic properties of the mutant enzymes were
examined. When the putative catalytic nucleophile Glu
was
changed to a Gln residue in the E231Q mutant, the specific activity was
reduced about 20,000-fold. At this level of activity, reliable
estimates of K
and k
could
not be obtained. The effect of mutating the postulated general acid
(Glu
) to a glutamine residue was less dramatic. The E288Q
mutant exhibited a 350-fold reduction in k
with
no significant change in K
(Table 1). Both
mutant enzymes exhibited bell-shaped pH profiles similar to that of the
wild-type MBP-GII (Fig. 3), although one might anticipate that
removal of either of the catalytic amino acids would alter the shape of
the pH rate curve dramatically. This observation raised the possibility
that the activity measured could be due to wt activity. The likelihood
of contamination of the expressed enzyme with wt enzyme appears to be
remote, given that individual cDNA clones were used in expression work.
Translational misreading is a possibility (Schimmel, 1989), but
spontaneous deamidation of the introduced Gln residue might be more
likely (Planas et al., 1992). To address these possibilities,
the thermal inactivation of the E288Q mutant was compared with that of
the wt enzyme (Fig. 7). The activity loss of the E288Q mutant
does not parallel that of the wt enzyme, and we therefore conclude that
the activity measured in the mutant preparation is due to the abundant,
low specific activity MBP-GII(E288Q) mutant enzyme itself and not due
to a small amount of wt enzyme with a high specific activity.
Figure 7:
The effect
of amino acid substitutions on the thermostability of MBP-GII.
Thermostabilities of MBP-GII(wt) (), MBP-GII(E288Q) (
),
MBP-GII(K282M) (
), and MBP-GII(E279Q) (
) were tested at
55 °C as described in the legend to Fig. 4.
The
relative impact on activity of mutations in the catalytic acid and the
catalytic nucleophile was unexpected and not in line with commonly
observed effects (Svensson and Søgård, 1993; Legler,
1993). Replacement of the general acid usually results in near complete
loss of activity, as observed for Tyr in
-galactosidase (Ring and Huber, 1990), Glu
in hen egg
white lysozyme (Malcolm et al., 1989), and
Glu
in Aspergillus niger glucoamylase (Sierks et al., 1990), while low to significant residual
activity usually remains after mutation of catalytic nucleophiles. For
example, the D52N mutation in hen egg white lysozyme, in which
Asp
is the nucleophile, reduces k
to 5% of wt activity on Micrococcus cells (Malcolm et al., 1989). In the case of MBP-GII, this trend is reversed.
Substitution of the nucleophile led to an almost complete loss of
activity, while significant levels of activity remained following
mutation of the catalytic acid. Nevertheless, similar results have been
recently recorded. The substitution of a Gln residue for the
nucleophilic Glu
in an Agrobacterium
-glucosidase (Withers et al., 1992) and
Glu
in a Bacillus licheniformis 1,3-1,4-
-glucanase (Planas et al., 1992)
resulted in dramatic loss of activity, while the substitution of a
potential, as yet unconfirmed, proton donor (Asp
) in the
glucosidase had a somewhat less dramatic effect (Trimbur et
al., 1992).
As expected for catalytic amino acid
residues, Glu and Glu
are highly conserved
in the primary structures of a wide range of eukaryotic 30-35-kDa
-glucanases (Chen et al., 1993a) (Fig. 5A). One exception is the anther-specific 53-kDa
protein encoded by the developmentally regulated Brassica napus and Arabidopsis thaliana A6 gene, which despite a 30%
sequence identity with isoenzyme GII is predicted to contain a
glutamine residue (Gln
) at the equivalent position of
Glu
in isoenzyme GII (Hird et al., 1993) (Fig. 5B). Significantly, to date, no glucanase
activity has been associated with the A6 protein when expressed
prematurely in tobacco anthers or in E. coli (Hird et
al., 1993). Therefore, these data support rather than undermine
the importance of Glu
and its equivalents for activity.
If the A6 protein is not active, what could the purpose of a glutamine
residue in place of the Glu
equivalent be? Since
premature expression of
-glucanase activity in anthers of
transgeneic tobacco results in male sterility (Worrall et al.,
1992), it is possible that A6 activity is either required to be very
low or alternatively regulated by deamidation. It has been previously
suggested that structure-dependent deamidation of proteins serve as in vivo molecular clocks that control development and aging
(Robinson and Robinson, 1991). Neighboring Thr and Ser residues have a
catalytic effect on Asn and Gln deamidation, and this may be why the
three least probable side-by-side residue pairs involving amides in
proteins are Gln-Ser, Asn-Thr, and Thr-Gln (Robinson and Robinson,
1991). We note that the B. napus A6 protein contains the
sequence Thr
-Gln
. Expression in
anthers of a mutated A6 gene (Q341E) could be most informative.
Earlier biochemical and
structural data did not implicate Glu in the catalytic
process, but its strict conservation between related eukaryotic
-glucanases and its location within one of the most conserved
stretches of primary structure nevertheless points to a crucial
function (see Fig. 5). The potential importance of Glu
was therefore tested through its mutation to the sterically
similar Gln residue. MBP-GII(E279Q) exhibited a 240-fold reduction in k
with a small increase in K
(Table 1), suggesting that a negative charge at this
position might be required for optimal enzyme activity. Because the
decrease in activity is comparable with that observed when the
catalytic acid is removed (mutant E288Q), Glu
might
influence the ionization status of Glu
. However, the
considerable distance between these residues suggests that the effect
would be most likely to be exerted through Lys
, which is
located between Glu
and Glu
(Fig. 6). The spatial relationship between these residues
in the wt enzyme is illustrated in Fig. S1. It is possible that
Glu
ensures Lys
exists in a protonated
state, thus allowing Glu
to retain its proton for
catalytic purposes (Fig. S1, panelA). In the
absence of Glu
, the long flexible side chain of
Lys
might promote the deprotonation of Glu
.
The conversion of Glu
to its carboxylate form through the
protonation of Lys
would therefore prevent protonation of
the glycosidic oxygen, and activity would be lost as a result (Fig. S1, panelB). Such a possibility could
be tested with a double mutant, MBP-GII(E279Q,K282M), only if
Lys
individually is non-essential for activity. To
explore this further, MBP-GII(K282M) was constructed and purified. The
use of methionine in place of lysine was based on the three-dimensional
structure, which indicated such a change would maintain the packing,
and van der Waals' contacts contributed by the four methylene
groups of the lysine residue in the wt enzyme. As shown in Table 1, a 2500-fold reduction in specific activity and a modest
increase in K
resulted from this mutation. Due to
the critical nature of the K282M mutation alone, a MBP-GII
(E279Q,K282M) double mutant could therefore not give critical
information in regard of our hypothesis and was therefore not
constructed.
Scheme 1:
Scheme 1Proposed mechanism of action of
isoenzyme GII and the effect of introduced mutations (see text for
details). Note the mechanism shown in this scheme occurs with retention
of configuration at the anomeric carbon, as has been deduced from
NMR.()
The results discussed above strongly indicate that a
tetrad of ionizable amino acids (Glu, Glu
,
Lys
, and Glu
) is of crucial importance for
the catalytic mechanism of this polysaccharide endohydrolase. While the
catalytic role of Glu
and Glu
now appears
well established, the mechanism(s) through which Glu
and
Lys
exert their influence remain(s) unclear at this
stage, although it is possible that these two residues form an ion pair
or a salt bridge, presenting Glu
with the positive side
of a dipole. This dipole may help stabilize the negative charge
developing on Glu
during protonation of the glycosidic
oxygen atom (Fig. S1A). Such a mechanism would be
analogous to that of T4 lysozyme, in which residues especially
sensitive to mutational alteration include those involved in buried
salt bridges near the catalytic site (in particular, a salt bridge
involving the presumed catalytic acid Glu
and Arg
(Rennel et al., 1991)). T4 lysozyme contains an
additional salt bridge between Asp
and His
,
which contributes to the enzyme's thermal stability, as judged
from the much lower T
of the D70N and H31N mutants
(Anderson et al., 1990). Similarly, the
MBP-GII(E279Q) and MBP-GII(K282M) mutants were significantly less
stable than wt MBP-GII (Fig. 7). While this points to the
presence of a salt bridge between these two residues, it is not certain
whether it actually contributes to the stability of the enzyme or
rather minimizes the potential instability caused by the introduction
of unmatched charges in the two mutant enzymes. However, the enhanced
stability of the E288Q mutation indicates that the overall number of
ionizable residues per se does not influence the stability and
again shows that the residual activities measured in the four mutant
enzymes are not due to contaminating wt-enzyme activity. It is
noteworthy that a similar enhancement of stability has been observed in
the E358Q mutant of an Agrobacterium
-glucosidase
(Withers et al., 1992).
To further investigate the
potential influence of Glu and Lys
on the
ionization state of Glu
, the pH-rate profiles of
MBP-GII(E279Q), MBP-GII(K282M), and wt MBP-GII were compared (Fig. 3). Small but significant differences were observed, and
these are consistent with the mechanisms presented in Fig. S1.
Thus, the pH optimum of MBP-GII(K282M) was clearly shifted toward
higher pH values, and the activity/pH profile broadened, consistent
with an increase of the pK
for Glu
in this mutant. The pH-rate profile of the E279Q mutant was
virtually identical to that of the wt enzyme, except for a small but
noticeable broadening of the pH-rate profile toward a more acidic pH.
Additional experimental evidence in support of the ionization state
of the residues shown in Fig. S1is the observation that
Glu but not Glu
is modified by
carbodiimide/glycine ethyl ester (Chen et al.,1993a)
and that Glu
sits in a relatively hydrophobic environment (Fig. 6), which could lead to an increase in the
pK
of the nearby Glu residue. It is possible that
the strategy employed by the glucanases is to raise the
pK
of Glu
through the positioning of
hydrophobic residues in its vicinity (Urry et al., 1993) and
at the same time to facilitate deprotonation due to the neighboring
dipole of Lys
and Glu
. Such an arrangement
may ensure that the proton on Glu
is exchanging
frequently, a prerequisite for catalysis. Such a delicate balance
between protonation states may well be influenced by the presence of
bound substrate.
Apart
from the nucleophilic action of Glu, the structural
analysis of the inhibitor-enzyme complex also supports other aspects of Fig. S1. The orientation of the inhibitor within the active site
indicates that substrate binding occurs with the non-reducing end
protruding toward the Western exit (as seen in Fig. 6A)
and the reducing end facing the Eastern exit. Furthermore, since the
length of the substrate binding cleft (40 Å) is sufficient to
accommodate about eight residues of an extended glucan chain and since
the two catalytic glutamic residues lie about one third of the way
along the cleft (as counted from the Western end in Fig. 6)
(Varghese et al., 1994), it appears that cleavage could occur
between the third and fourth glucosyl binding subsite (numbering from
the non-reducing end of the substrate), a conclusion that has been
supported by independent mapping of binding subsites by kinetic
analysis. (
)Excitingly, the refined structure also clearly
indicates the presence of a water molecule bound via a hydrogen bond to
the catalytic acid (Glu
). The positioning of such a
molecule would allow it to regenerate the active enzyme following
release of the aglycon, as outlined in panelA of Fig. S1. Finally, the glucose moiety is hydrogen bonded via O-6
to both Glu
and Lys
. Thus, while
Glu
and Lys
, as discussed above, may well
enhance catalytic efficiency of MBP-GII through their influence on the
ionization state of Glu
, this is unlikely to constitute
their only role. Indeed, the fact that MBP-GII(E288Q) exhibits 7-fold
higher activity than MBP-GII(K282M) in particular points to additional
crucial roles for Lys
, for example, binding of substrate.
Such a role would be consistent with the 240% increase in K
seen of MBP-GII(K282M) for the polymeric
laminarin substrate. Therefore, in the absence of Lys
,
substrate bound at the active site cleft may be positioned in a
sub-optimal fashion in relation to the catalytic nucleophile and the
catalytic acid, thus leading to a substantial loss of activity (Fig. S1C) (see also Høj et al. (1989b)). The sub-optimal binding could be due to an effect on the
substrate-enzyme interaction only and/or to a change in active site
conformation induced by the absence of the putative
Glu
-Lys
salt bridge. We cannot
discriminate between these possibilities at present, but note that a
cluster of ionizable amino acids also is found in the
-amylases,
which invariably contains Asp
, Glu
, and
Asp
(barley
-amylase numbering) at the active site.
As seen for the
-glucanases,
-amylase catalysis requires all
three groups, and stabilization of the transition state is mediated by
the neighboring and invariant His
, which is believed to
hydrogen bond to the 6-OH of the substrate glycon ring adjacent to the
cleavage site (Søgaard et al., 1993). Further studies
are needed to establish whether such clusters of charged amino acids in
distinct classes of enzymes reflect a basic requirement for
polysaccharide hydrolysis.