Protein Engineering vol.11 no.2 pp.127–133, 1998
Mutations to alter Aspergillus awamori glucoamylase selectivity.
II. Mutation of residues 119 and 121
Tsuei-Yun Fang, Richard B.Honzatko1, Peter J.Reilly2
and Clark Ford3
Departments of Food Science and Human Nutrition, 1Biochemistry and
Biophysics and 2Chemical Engineering, Iowa State University, Ames, IA
50011, USA
3To
whom correspondence should be addressed
Mutations Ser119→Glu, Ser119→Gly, Ser119→Trp,
Gly121→Ala and Gly121→Ala/Ser411→Gly were constructed in glucoamylase to change substrate specificity.
Mutation Ser411→Gly was already known to decrease
glucoamylase selectivity toward isomaltose formation and
to increase peak glucose yield. All mutated glucoamylases
had slightly lower specific activities on maltose than on
wild-type glucoamylase. Ser119→Glu, Ser119→Gly and
Ser119→Trp glucoamylases were about as active on
isomaltose and DP 4–7 maltooligosaccharides as wild-type
glucoamylase. Gly121→Ala and Gly121→Ala/Ser411→Gly
glucoamylases were less active. At 55°C Ser119→Glu,
wild-type, Ser119→Trp, Ser119→Gly, Gly121→Ala and
Gly121→Ala/Ser411→Gly glucoamylases had progressively higher peak glucose yields, generally in the opposite
order to their activities. There was also an inverse
correlation between peak glucose yield and ratio of initial
rate of isomaltose production from glucose condensation
to that of glucose production from maltodextrin hydrolysis. The effect of mutations Gly121→Ala and
Ser411→Gly was not additive in predicting the effect of
the double mutation on the ratio or on peak glucose yield.
Keywords: glucoamylase/glucose condensation/glucose yield/
isomaltose/selectivity/site-directed mutagenesis/substrate specificity
Introduction
Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3,
GA) catalyzes the release of β-D-glucose from the nonreducing
ends of starch and related oligo- and polysaccharides. However,
at the high substrate concentrations used in industry, reversion
products, especially isomaltose [α-D-glucopyranosyl-(1,6)D-glucose, iG2], produced from glucose condensation limit Dglucose yield to about 95%. This suggests that engineering
GA substrate specificity to decrease the ability of GA to
synthesize the α-1,6-glucosidic bonds found in iG2 would
increase the D-glucose yield.
The GA catalytic mechanism involves two carboxyl groups
(Hiromi et al., 1966), Glu179 and Glu400 in Aspergillus
awamori or Aspergillus niger GA numbering (Sierks et al.,
1990; Harris et al., 1993; Frandsen et al., 1994; Ohnishi et al.,
1994). Glu179 protonates the oxygen in the glycosidic linkage,
acting as a general acid catalyst and Glu400 activates water
for nucleophilic attack at carbon C-1, acting as a general base
catalyst (Frandsen et al., 1994). The crystal structures of GA
complexed with the pseudotetrasaccharides acarbose and D© Oxford University Press
gluco-dihydroacarbose show that they have two different
binding conformers, A and B, at pH 4 (Figure 1) but only one
at pH 6 (conformer B) (Aleshin et al., 1994, 1996; Stoffer
et al., 1995). At pH 4, conformer A is the more prevalent
(53% with acarbose and 63% with D-gluco-dihydroacarbose).
Although subsite mapping indicates that the GA active site
has about six subsites (Hiromi et al., 1973, 1983; Tanaka
et al., 1983; Meagher et al., 1989), the pH-dependent interaction
of pseudotetrasaccharides suggests that only the first two
subsites are common to maltooligosaccharides and their substrate analogues (Aleshin et al., 1996).
In this study, site-directed mutagenesis was used to construct
mutations at residues 119 and 121 to alter hydrogen bonding
between enzyme and substrate. These residues have already
been subjected to site-directed mutagenesis in an attempt
to change substrate specificity by decreasing the rate of
condensation reactions to form α-1,6-glucosidic bonds. Sierks
and Svensson (1994) reported that the Ser119→Tyr mutation
increased the ratio of catalytic efficiency for hydrolysis of
maltose [α-D-glucopyranosyl-(1,4)-D-glucose, G2] to that of
iG2 (the selectivity ratio) 3.5-fold compared with wild-type
GA. However, Svensson et al. (1995) later showed that this
mutant GA has an unusually high capacity to produce branched
oligosaccharides under conditions similar to those in industrial
saccharification. Residue Gly121 has been mutated to Thr
(Natarajan and Sierks, 1996), decreasing the selectivity ratio
fourfold compared with wild-type GA.
The mutations reported in this paper were also designed
to change substrate specificity by decreasing the rate of
condensation reactions to form α-1,6-glucosidic bonds while
maintaining wild-type ability to hydrolyze α-1,4-linked substrates. Atom OG of Ser119 hydrogen bonds to the 3-OH of
the fourth glucosyl residue from the non-reducing end only in
conformer B, while the amide nitrogen of Gly121 hydrogen
bonds to the 6-OH of the third glucosyl residue in both
conformers A and B (Figure 1). Ser119 is not conserved but
is replaced by Ala, Pro, Glu, Asp, Arg and Tyr in other
GAs (Coutinho and Reilly, 1997). Mutation Ser119→Glu was
designed to strengthen the hydrogen bond between GA and
the fourth glucosyl residue to stabilize conformer B and to
add a negative charge near subsite 4 in order to increase
electrostatic interactions in the active site. The Ser119→Gly
mutation was designed to remove the same hydrogen bond in
order to destabilize conformer B. Mutation Ser119→Trp was
meant to remove that hydrogen bond and to increase hydrophobic interactions between the enzyme and conformer B.
Gly121 is highly conserved in all GA sequences except
Clostridium sp. G0005 and Methanococcus jannaschii GA,
where it is replaced by Thr (Coutinho and Reilly, 1997). The
former has high α-1,6-hydrolytic activity (Ohnishi et al.,
1992). Since the φ and ψ angles of Gly121 would allow Ala
in this position without causing a conformation distortion, the
Gly121→Ala mutation was designed to introduce a β-carbon
at position 121 to displace the 6-OH group of the third glucosyl
127
T.-Y.Fang et al.
Fig. 1. Stereo view of the active site of GA from Aspergillus awamori var. X100 (Stoffer et al., 1995) complexed with D-gluco-dihydroacarbose (bold).
Subsites 1–4 bind the D-gluco-dihydroacarbose residues from the top to the bottom, respectively, of the active site. Dashed lines represent hydrogen bonds.
The catalytic water is Wat 500.
residue from its hydrogen bonding position. In addition, the
double mutation Gly121→Ala /Ser411→Gly was designed to
investigate additivity of two mutations that increased substrate
specificity. The Ser411→Ala and Ser411→Gly mutations were
already known to increase the peak glucose yield from maltodextrin hydrolysis and to decrease significantly the ratio of
initial rate of iG2 formation from glucose condensation to that
of glucose formation from maltodextrin hydrolysis compared
with wild-type GA (Fang et al., 1998); the latter was combined
with the Gly121→Ala mutation because it allowed much more
activity to be maintained than the Ser411→Ala mutation.
Fang et al. (1998) also made Tyr116→Trp, Tyr175→Phe and
Arg241→Lys mutations in GA to affect substrate specificity
and achieved higher peak glucose yields with all of them.
They reported that ratios of initial rate of iG2 formation to
that of glucose formation better predicted peak glucose yields
than did selectivity ratios.
Materials and methods
Materials, GA production and purification, protein concentration measurement, enzyme kinetics, hydrolysis of DE (dextrose
equivalent) 10 maltodextrin and glucose condensation reactions
were as described by Fang et al. (1998). As before, the GA
concentration was 2.64 µM for all experiments unless stated
otherwise.
Construction of mutant GA genes
The GA gene was subjected to site-directed mutagenesis by
the protocols of either the Bio-Rad Muta-Gene phagemid
in vitro mutagenesis kit (Fang et al., 1998) or the Promega
Altered Sites II in vitro mutagenesis system. An XbaI–HindIII
fragment of pGEM7-GA containing the wild-type GA cDNA
(Fang et al., 1998) was inserted into the Promega pALTER-1
vector to make a GA cDNA-containing vector to be used as
the double-stranded DNA template in the Altered Sites II
system. The following mutation-containing oligonucleotide
primers were synthesized at the Iowa State University Nucleic
Acid Facility: 59-GCC TAC ACT GGT GAA TGG GGA CGG
CC-39 (Ser119→Glu), 59-GCC TAC ACT GGT GGA TGG
GGA CGG CC-39 (Ser119→Gly), 59-CC TAC ACT GGT
TGG TGG GGA CGG CC-39 (Ser119→Trp) and 59-ACT
GGT TCT TGG GCT CGG CCG CAG C-39 (Gly121→Ala).
Nucleotides for designed GA mutations are shown in bold.
The double mutation Gly121→Ala/Ser411→Gly was constructed by ligating an XhoI–PstI fragment carrying the
128
Table I. Specific activities (IU/mg enzyme) of wild-type and mutant GAsa
GA form
35°C
Wild-type
Ser119→Glu
Ser119→Gly
Ser119→Trp
Gly121→Ala
Gly121→Ala/Ser411→Gly
7.8
6.8
5.3
8.2
6.4
5.9
6
6
6
6
6
6
0.1b
0.1
0.2
0.1
0.2
0.2
45°C
55°C
17.0 6 0.1
13.5 6 0.4
11.8 6 0.1
15.8 6 0.5
14.8 6 0.3
14.3 6 0.3
31.2
26.8
22.3
29.9
24.1
23.8
6
6
6
6
6
6
2.1
0.7
0.6
0.3
0.3
0.7
aDetermined
from the hydrolysis of 0.0974 M G2 in 0.05 M NaOAc buffer
at pH 4.4.
bStandard error.
Gly121→Ala mutation and a PstI–BamHI fragment carrying
the Ser411→Gly mutation (Fang and Ford, 1998) to the
XhoI–BamHI fragment of YEpPM18 to reconstruct the yeast
expression vector YEpPM18. For single mutations, the mutated
GA cDNAs were subcloned into YEpPM18 as described
previously (Chen et al., 1994). All designed mutations were
verified by DNA sequencing of the entire subcloned fragment.
The mutated YEpPM18 was transformed into Saccharomyces
cerevisiae C468 by electroporation.
Specific activity assays
Specific activity assays were performed at 35, 45 and 55°C
with 0.0974 M G2 in 0.05 M NaOAc buffer at pH 4.4.
Reactions were stopped at six different times by adding
samples to 0.4 volume of 4 M Tris–HCl buffer, pH 7.0, and
the glucose concentration was measured by the glucose oxidase
method (Rabbo and Terkildsen, 1960). Average values of
duplicated experiments were used. One unit (IU) is defined as
the amount of enzyme required to produce 1 µmol of glucose
per minute under the conditions of the assay.
Results
Specific activity of GA at different temperatures
Specific activities of mutated GAs were in general slightly
below those of wild-type GA (Table I), being lowest (about
70% of wild-type values) in Ser119→Gly GA. Activation
energies ranged from 54.4 kJ/mol (Ser119→Trp GA) to
60.5 kJ/mol (Ser119→Gly GA), with that of wild-type GA
being 58.3 kJ/mol, indicating that changes of temperature
affected activities of different mutated GAs similarly and that
these mutations did not change the catalytic mechanism.
Mutations to alter glucoamylase selectivity
Table II. Kinetic parameters of wild-type and mutant GAs for hydrolysis of iG2 and G2–G7 at 45°C in 0.05 M NaOAc buffer, pH 4.4
GA form
iG2
Wild-typea
kcat (s–1)
0.72 6 0.01b
KM (mM)
23.5 6 0.6
kcat/KM (s–1 mM–1) 0.031 6 0.001
660
Selectivity ratioc
Ser119→Glu
kcat (s–1)
0.91 6 0.04
28.5 6 3.4
KM (mM)
kcat/KM (s–1 mM–1) 0.032 6 0.003
Selectivity ratio
420
∆(∆G)d (kJ/mol)
–0.11
Ser119→Gly
0.53 6 0.01
kcat (s–1)
KM (mM)
12.2 6 1.2
kcat/KM (s–1 mM–1) 0.043 6 0.004
Selectivity ratio
440
∆(∆G) (kJ/mol)
–0.89
Ser119→Trp
kcat (s–1)
0.94 6 0.03
KM (mM)
32.7 6 2.4
–1
–1
kcat/KM (s mM ) 0.029 6 0.001
Selectivity ratio
460
∆(∆G) (kJ/mol)
0.16
Gly121→Ala
kcat (s–1)
0.58 6 0.02
25.1 6 2.6
KM (mM)
kcat/KM (s–1 mM–1) 0.023 6 0.002
Selectivity ratio
1210
∆(∆G) (kJ/mol)
0.75
Gly121→Ala/Ser411→Gly
0.47 6 0.02
kcat (s–1)
38.8 6 4.2
KM (mM)
kcat/KM (s–1 mM–1) 0.012 6 0.001
Selectivity ratio
1480
∆(∆G) (kJ/mol)
2.45
Ser411→Glya
kcat (s–1)
0.93 6 0.06
KM (mM)
26.2 6 2.7
kcat/KM (s–1 mM–1) 0.036 6 0.004
Selectivity ratio
400
∆(∆G) (kJ/mol)
–0.39
aData from Fang
bStandard error.
cSelectivity
dChange of
G2
G3
G4
G5
G6
G7
20.4 6 0.2
1.01 6 0.03
20.3 6 0.6
48.2 6 0.7
0.25 6 0.01
196 6 9
64.5 6 2.9
0.111 6 0.017
582 6 65
71.8 6 1.9
0.110 6 0.010
654 6 43
73.7 6 2.1
0.107 6 0.010
685 6 47
72.3 6 0.9
0.083 6 0.004
870 6 35
16.5 6 0.5
1.21 6 0.12
13.5 6 1.0
41.4 6 0.5
0.32 6 0.01
129 6 4
63.0 6 1.4
0.164 6 0.011
385 6 19
67.6 6 0.7
0.098 6 0.004
691 6 23
65.9 6 0.8
0.079 6 0.004
832 6 36
94.4 6 1.8
0.107 6 0.007
883 6 48
1.07
1.11
14.1 6 0.4
0.75 6 0.09
18.7 6 1.8
38.4 6 0.9
0.26 6 0.03
151 6 12
0.21
0.69
0.63
19.0 6 0.7
1.43 6 0.16
13.3 6 1.1
45.0 6 0.7
0.48 6 0.02
94.6 6 3.4
47.7 6 2.3
0.27 6 0.03
180 6 14
59.8 6 1.2
0.23 6 0.01
263 6 10
66.0 6 3.3
0.28 6 0.03
235 6 18
88.0 6 1.6
0.25 6 0.01
346 6 10
1.11
1.93
3.10
2.41
2.83
2.44
17.8 6 0.2
0.64 6 0.04
27.9 6 1.4
32.7 6 0.6
0.58 6 0.03
56.8 6 2.3
62.8 6 1.1
0.61 6 0.03
102 6 3
48.7 6 0.6
0.68 6 0.02
71.8 6 1.5
51.2 6 0.9
0.79 6 0.03
65.2 6 1.6
49.3 6 1.2
0.67 6 0.004
73.7 6 2.3
–0.85
3.28
4.60
5.84
6.22
6.53
17.8 6 0.4
1.00 6 0.07
17.8 6 1.0
38.1 6 0.9
1.29 6 0.08
29.6 6 1.4
59.3 6 1.3
1.05 6 0.06
56.6 6 2.0
63.4 6 2.2
1.78 6 0.16
35.5 6 2.1
61.1 6 2.1
1.78 6 0.16
34.3 6 2.0
55.1 6 0.8
1.34 6 0.06
41.0 6 1.2
0.34
5.00
6.17
7.72
7.92
8.08
23.0 6 0.4
1.59 6 0.08
14.5 6 0.6
55.1 6 1.6
0.50 6 0.04
108 6 6
59.7 6 1.8
0.092 6 0.010
649 6 55
75.1 6 2.1
0.094 6 0.010
795 6 61
0.89
1.56
–0.29
–0.52
1.09
72.8 6 2.2
0.159 6 0.015
458 6 30
–0.14
75.7 6 1.6
0.111 6 0.008
680 6 39
–0.10
–0.51
81.1 6 2.4
0.124 6 0.012
652 6 50
0.13
75.9 6 4.3
0.125 6 0.024
609 6 87
0.31
–0.04
124 6 4
0.112 6 0.013
1110 6 100
–0.64
84.0 6 2.5
0.132 6 0.012
634 6 41
0.84
et al. (1998).
ratio: [kcat/KM(G2)]/[kcat/KM(iG2)].
transition-state energy. ∆(∆G)5 –RTln[(kcat/KM)mut/(kcat/KM)wt] (Wilkinson et al., 1983).
Enzyme kinetics
Kinetic parameters (kcat and KM) for the hydrolysis of α-1,6linked iG2 and α-1,4-linked G2, maltotriose (G3), maltotetraose
(G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) at 45°C and pH 4.4 are given in Table II.
Values of kcat for G2 and G3 were somewhat lower for
mutant GAs than for wild-type GA, agreeing with the specific
activity results above. Except for Gly121→Ala GA, which
had low kcat values for substrates of all chain lengths, values
of other mutant GAs approached or even exceeded those of
wild-type GA for G4–G7. Values of KM were roughly the same
for Ser119→Glu and Ser119→Gly GAs as for wild-type GA;
they were significantly higher for Ser119→Trp, Gly121→Ala
and especially for Gly121→Ala/Ser411→Gly GAs. This led
to catalytic efficiencies (kcat/ KM) that were lower than wildtype GA values at lower chain-length substrates for Ser119→
Glu and Ser119→Gly GAs and were about the same at higher
chain lengths. Other mutated GAs generally had lower catalytic
efficiencies with G2–G7. Resulting ∆(∆G) values were similar
to those of wild-type GA for Ser119→Glu and Ser119→Gly
GAs and progressively more positive for Ser119→Trp, Gly121
→Ala and Gly121→Ala/Ser411→Gly GAs.
Mutations Gly121→Ala and Gly121→Ala/Ser411→Gly
increased the selectivity ratios about twofold compared with
wild-type GA. Mutations Ser119→Glu, Ser119→Gly and
Ser119→Trp had decreased selectivity ratios.
129
T.-Y.Fang et al.
Table III. Initial rates of glucose and iG2 formation in the hydrolysis of
28% (w/v) DE 10 maltodextrin and condensation of 30% (w/v) glucose,
respectively, and their relative ratios for wild-type and mutant GAs at 35
and 55°C in 0.05 M NaOAc buffer at pH 4.4
GA form
Initial rate (mol/mol GA·s)
Glucose
35°C
Wild-typea
Ser119→Glu
Ser119→Gly
Ser119→Trp
Gly121→Ala
Gly121→Ala/Ser411→Gly
Ser411→Glya
34.7
28.6
37.2
39.7
30.0
28.3
30.0
55°C
Wild-typea
Ser119→Glu
Ser119→Gly
Ser119→Trp
Gly121→Ala
Gly121→Ala/Ser411→Gly
Ser411→Glya
226 6 19
191 6 16
204 6 10
193 6 13
121 6 9
111 6 6
154 6 8
aData
6
6
6
6
6
6
6
1.9b
1.9
2.2
1.9
2.2
1.9
1.9
iG23103
(Initial rate
iG2/initial rate
Glc) 3103
16.4
14.3
15.1
12.9
8.28
4.31
6.06
6
6
6
6
6
6
6
0.8
0.4
0.5
0.5
0.50
0.22
0.86
0.47
0.50
0.41
0.32
0.28
0.15
0.20
120
124
102
103
62.5
25.9
31.4
6
6
6
6
6
6
6
5
7
3
4
1.7
0.6
2.2
0.53
0.65
0.50
0.54
0.52
0.23
0.20
from Fang et al. (1998).
error.
bStandard
Fig. 2. Formation of glucose by wild-type and mutant GAs during
hydrolysis of 28% (w/v) DE 10 maltodextrin in 0.05 M NaOAc buffer,
pH 4.4, and 0.02% sodium azide for 12 days at (a) 35 and (b) 55°C.
(s) Wild-type, (u) Ser119→Glu, (n) Ser119→Gly, (.) Ser119→Trp, (r)
Gly121→Ala, (A) Gly121→Ala/Ser411→Gly.
Hydrolysis of DE 10 maltodextrin
DE 10 maltodextrin hydrolysis was used to study glucose
formation by wild-type and mutant GAs at high substrate
concentrations in order to simulate industrial saccharification
(Figure 2). Initial rates of glucose formation are given in
Table III.
At 35°C, glucose yields from maltodextrin hydrolysis were
apparently still increasing with increasing times for all GAs,
but were much higher at any time for wild-type, Ser119→Glu,
Ser119→Gly and Ser119→Trp GAs than for Gly121→Ala
and Gly121→Ala/Ser411→Gly GAs (Figure 2a). Initial rates
of glucose formation for Ser119→Trp, Ser119→Gly and wildtype GAs were higher than those for Gly121→Ala, Ser119→
Glu and Gly121→Ala/Ser411→Gly GAs (Table III). For
Gly121→Ala and Gly121→Ala/Ser411→Gly GAs, GA concentrations of 3.30 and 3.96 µM were used in addition to
2.64 µM for maltodextrin hydrolysis (data not shown), leading
to proportional increases in initial rates of glucose formation
130
but to only very small increases in glucose yield at higher
incubation times.
At 55°C, Ser119→Glu, Ser119→Gly, Ser119→Trp and
especially wild-type GAs reached high glucose yields very
quickly, whereas the glucose yields of Gly121→Ala and
Gly121→Ala/Ser411→Gly GAs slowly increased over a long
period, eventually reaching higher levels than the others (Figure
2b). All mutant GAs had lower initial rates of glucose
formation than wild-type GA, Gly121→Ala and Gly121→Ala/
Ser411→Gly GAs being especially low (Table III). Initial rates
for wild-type and Ser119→Glu GAs at 55°C increased about
sevenfold over those at 35°C, whereas they increased only
about four- to sixfold for the other mutant GAs, suggesting
that changes in temperature had a more varied effect on rates
of glucose formation from maltodextrins by different mutated
GAs than they did on specific activity on G2.
Glucose condensation reactions
Glucose condensation reactions were used to study the ability
of wild-type and mutant GAs to synthesize iG2 at high glucose
concentrations (Figure 3). Initial rates of iG2 formation are
given in Table III.
At 35°C, all mutant GAs had lower initial rates of iG2
formation than wild-type GA, with rates being especially low
for Gly121→Ala and Gly121→Ala/Ser411→Gly GAs (Figure
3a and Table III). Initial rates of iG2 formation at 55°C
increased six- to ninefold over those at 35°C and were lower
than wild-type GA for all other GAs except Ser119→Glu GA.
Changes of temperature generally appeared to have a greater
effect on the rate of iG2 formation than on that of glucose
formation by different GAs.
Selectivity of GA for α-1,6-linked product synthesis versus
α-1,4-linked substrate hydrolysis
The ratio of the initial rate of iG2 formation from glucose
condensation to that of glucose formation from maltodextrin
hydrolysis was used to indicate the selectivity for α-1,6linked product synthesis vs α-1,4-linked substrate hydrolysis.
Mutations to alter glucoamylase selectivity
Fig. 4. Relationship between peak glucose yields and (initial rate of iG2
formation/initial rate of glucose formation) at 55°C in the incubation of
28% (w/v) DE 10 maltodextrin and 30% (w/v) D-glucose, respectively, in
0.05 M NaOAc at pH 4.4 with 0.02% sodium azide for 12 days with wildtype and mutant GAs.
Fig. 3. Formation of iG2 during incubation of 30% (w/v) D-glucose in
0.05 M NaOAc buffer at pH 4.4 with 0.02% sodium azide for 12 days at
(a) 35 and (b) 55°C with wild-type and mutant GAs. Symbols as in
Figure 2.
Ser119→Gly, Ser119→Trp, Gly121→Ala and Gly121→Ala/
Ser411→Gly GAs, especially the last, had lower ratios than
wild-type GA at 35°C, while Ser119→Glu GA had a similar
ratio (Table III). At 55°C, Ser119→Glu GA had a higher ratio
than wild-type GA, while Ser119→Gly, Ser119→Trp and
Gly121→Ala GAs had very similar ratios, and Gly121→Ala/
Ser411→Gly GA still had a much lower ratio. Ratios of all
GAs, especially those of Ser119→Trp, Gly121→Ala and
Gly121→Ala/Ser411→Gly GAs, were higher at 55 than at
35°C.
Since maltodextrin hydrolysis and glucose condensation
were performed in the same buffer with the same amount
of GA and almost the same substrate concentrations, these
reactions were compared. Because maltodextrin hydrolysis at
35°C did not reach completion, only data obtained at 55°C
were used. Peak glucose yields and ratios of initial rates of
iG2 formation to those of glucose formation had an inverse
relationship (Figure 4).
Discussion
Residues 119 and 121 were mutated to study the effect of
hydrogen bonding between GA and substrate on substrate
specificity. The latter was evaluated not only from kinetic
parameters (Table II) but also from formation of iG2 and glucose
using highly concentrated glucose and DE 10 maltodextrin,
respectively, as substrates (Table III).
The kinetic parameters of Ser119→Glu GA showed that
this mutation only slightly altered the active site. Catalytic
efficiency on short-length substrates, however, was affected
more than on long-length substrates. This was possibly due to
increased electrostatic interactions from the introduced negative
charge near subsite 4 affecting hydrolysis of short-length
substrates.
Although mutation Ser119→Gly was expected to remove
the hydrogen bond between residue 119 and the fourth substrate
residue to destabilize conformer B, observed changes in ∆(∆G)
did not confirm this. Fersht (1985) reported that in certain
cases, such as a deletion of an enzyme side-chain allowing
the access of water into the enzyme–substrate complex, loss
of a hydrogen bond may cause no loss of binding energy. In
fact, removal of the Ser119 side-chain by the Ser119→Gly
mutation potentially allows a water molecule to enter the space
between residue 119 and the substrate. The Ser119→Gly
mutation increased kcat values for G4–G7 hydrolysis whereas
it decreased them for iG2, G2 and G3 hydrolysis compared
with wild-type GA. KM values decreased only for iG2 and G2
hydrolysis. These lower kcat and KM values indicate that the
Ser119→Gly mutation affected subsites 1 and 2. Since this
mutation altered the flexibility of a peptide bond near Trp120,
it may affect the side-chain position of Trp120. Trp120 is an
important residue, as shown by the effects caused by mutating
it (Sierks et al., 1989) and by three-dimensional structures of
GA complexed with pseudotetrasaccharides (Stoffer et al.,
1995; Aleshin et al., 1996). Atom NE1 of Trp120 hydrogen
bonds to atom OE2 of Glu179, the catalytic acid and atoms
CE3, CZ3 and CD2 of Trp120 make non-bonded contacts with
atom O5 of the third sugar residue of pseudotetrasaccharide
inhibitors (Figure 1). Sierks et al. (1989) reported that mutation
of Trp120 to His, Leu, Phe and Tyr decreased kcat and KM
131
T.-Y.Fang et al.
values for G2 hydrolysis and they also suggested that Trp120
is important for stabilization of the transition-state enzyme–
substrate complex in subsites 1 and 2.
Mutation Ser119→Trp was designed to remove the same
hydrogen bond as mutation Ser119→Gly and to increase
hydrophobic interactions between the enzyme and conformer
B. ∆(∆G) values for G4–G7 hydrolysis by Ser119→Trp GA,
2.4–3.1 kJ/mol, indicated that the mutation caused the loss of
a hydrogen bond between an uncharged GA group and the
substrate, probably at the fourth subsite. Increased hydrophobic
interactions near subsite 4 might have affected subsites 1 and
2, as shown by our previous study on Tyr175→Phe GA (Fang
et al., 1998).
Mutation Gly121→Ala was designed to introduce a βcarbon at position 121 to displace the 6-OH group of the third
sugar residue from its hydrogen bonding position. ∆(∆G)
values indicated that the hydrogen bond between residue 121
and the third sugar residue of the substrate had been removed,
which is likely responsible for the increased KM values for
G3–G7 hydrolysis. The Gly121→Ala mutation also decreased
the catalytic efficiency for iG2 hydrolysis and increased it for
G2 hydrolysis, doubling the selectivity ratio over that of wildtype GA. Since the mutation also affects the flexibility of the
peptide bond next to Trp120, it might also have affected the
side-chain position of Trp120 and therefore subsites 1 and 2
as in Ser119→Gly GA.
The double mutation Gly121→Ala/Ser411→Gly was
designed to investigate additivity of two GA mutations meant
to change substrate specificity. Values of kcat for the double
mutant GA were roughly the average of those for the two
single mutant GAs, except for iG2 (Table II). However, KM
values of the double mutant GA were higher than the
already high values of Gly121→Ala GA and bore no
resemblance to those of Ser411→Gly GA. Consequently, the
selectivity ratio of the double mutant GA was somewhat higher
than the high ratio of Gly121→Ala GA and much different to
the low ratio of Ser411→Gly GA. Furthermore, ratios of initial
rate of iG2 formation to that of glucose formation were very
low for Gly121→Ala/Ser411→Gly GA, similar to those of
Ser411→Gly GA but substantially below those of Gly121→Ala
GA (Table III). Peak glucose yield of the double mutant GA
at 55°C was higher than that of either single mutant GA
(Figure 4) (Fang et al., 1998). Unfortunately, it was even less
active than either of them. It appears, therefore, that the
properties of Gly121→Ala/Ser411→Gly GA were a complicated mixture of the properties of each of the two GAs
possessing its mutations.
Gly121→Ala and Gly121→Ala/Ser411→Gly GAs produced
glucose very slowly from maltodextrins as hydrolysis progressed, especially at 35°C (Figure 2a), even when higher
enzyme concentrations were used. This appears to have been
caused by the exceedingly high values of KM and low values
of kcat/KM for longer maltooligosaccharides compared with
other GAs, which would cause them to remain unhydrolyzed
even at long reaction times.
Changes in temperature played a complicated role in the
measured properties of wild-type and mutant GAs. Increased
temperatures increased specific activities of different GAs on
G2 almost equally, giving very similar activation energies.
However, initial rates of iG2 formation increased relatively
more than those of glucose formation with increased
temperature, yielding higher ratios of the two rates at higher
temperatures. Furthermore, rates of glucose formation
132
increased differently for different GAs, increasing ratios for
Gly121→Ala, Ser119→Trp and Gly121→Ala/ Ser411→Gly
GAs more than for the others. These increased ratios could be
caused by the removal of hydrogen bonds between mutated
GAs and the third and fourth glucosyl residues from the nonreducing end, which was the desired outcome of all but the
Ser119→Glu mutation. This would lead to greater loss of GA
binding at higher temperatures on the longer-length substrates
whose hydrolysis yields glucose than loss of binding on
disaccharides like G2 and iG2.
Conclusion
GA substrate specificity has been changed by altering hydrogen
bonding between enzyme and substrate at two positions in the
enzyme–substrate complex. This led to progressively higher
peak glucose yields from maltodextrin hydrolysis for
Ser119→Trp, Ser119→Gly, Gly121→Ala and Gly121→Ala/
Ser411→Gly GAs at 55°C but to generally lower activities
for all mutant GAs, decreases in activity being more or less
correlated with increased yield. As found earlier (Fang et al.,
1998), ratios of initial rates of iG2 formation from glucose to
that of glucose formation from maltodextrin were a better
predictor of peak glucose yield than were selectivity ratios.
This would be expected with these mutations, since residues
119 and 121 interact with the fourth and third substrate residues,
respectively, which are found in maltooligosaccharides but
not in G2 and iG2. Most important, a rational basis has now
been established for the mutation of GA to obtain higher
glucose yields from maltodextrin hydrolysis.
Acknowledgments
This project was supported by the US Department of Energy through
the Consortium for Plant Biotechnology Research, the US Department of
Agriculture through Grant 95-37500-1926 and through the Midwest Advanced
Food Manufacturing Alliance and by Genencor International. We thank Dr
James Meade for the gift of wild-type GA gene and plasmid and Drs John
F.Robyt and Motomitsu Kitaoka for help with iG2 quantitative determination
by thin-layer chromatography. This is Journal Paper No. J-17576 of the Iowa
Agriculture and Home Economics Experiment Station, Ames, IA, Project No.
3420, supported by Hatch Act and State of Iowa funds.
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