SUPPLEMENTARY ONLINE DATA
Mechanism of rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis
Jürgen JALAK and Priit VÄLJAMÄE
Institute of Molecular and Cell Biology, University of Tartu, Vanemuise 46 – 138, 51014 Tartu,
Estonia
Figure 1 (A) Hydrolysis of cellulose by CBH with (, , ) and without (, , ) the presence of
0.5 mM pNPL.  and , 2.5 μM PcCel7D, PWS 1.0 g l-1;  and , 2.5 μM TrCel7A, PWS 1.0 g l-1;
 and , 0.5 μM PcCel7D CD, PWS 2.5 g l-1. (B) Hydrolysis of 0.5 mM pNPL by 0.85 μM BG. This
activity was not dependent on the type and concentration of cellulose present. Error bars are from 7
independent measurements. (C) Rate of the hydrolysis of pNPL by CBH in the presence of cellulose
and BG at different concentrations. 0.5 mM pNPL, 2.5 μM TrCel7A, Avicel 10 g l-1. BG was , 0
μM; , 0.73 μM; , 0.85 μM; , 1.46 μM. (D) Hydrolysis of cellulose by CBH with (, ) and
without (, ) the presence of 0.85 μM BG.  and , 2.5 μM TrCel7A, Avicel 10 g l-1;  and ,
1.0 μM TrCel7A, PWS 10 g l-1. (E) All-free lines represent the hydrolysis of pNPL by CBH in the
absence of cellulose. pNPL was 0.5 mM. In coordinates (vpNP/[CBH]) versus [pNP] data points
obtained at CBH concentrations 0.5 - 2.5 μM fall in the same line. Solid lines represent non-linear
regression according to the Equation (10). The values of kcat(pNPL), KM(pNPL) and Ki(Lac) of 5.25 min-1, 500
μM and 80 μM respectively were obtained for TrCel7A. Corresponding figures for PcCel7D were 4.0
1
min-1, 1000 μM and 36.7 μM. These figures were used in calculation of [CBH]FA from vpNP measured
in the presence of cellulose according to the Equation (11) (see also supplementary Table I). ,
TrCel7A; , PcCel7D. (F) Binding kinetics of TrCel7A to Avicel. Total bound CBH ([CBH]bound, 
and ) was found from the binding experiment as difference between total CBH and CBH free from
cellulose. CBH with occupied active site ([CBH]OA,  and ) was found from parallel experiment
according to the Equation (1). Avicel was 10 g l-1 and TrCel7A was 2.5 μM ( and ) or 1.0 μM (
and ).
Table I. Calculation of observed catalytic constants (kobs)a for CBH catalyzed hydrolysis of
cellulose
Time
min
[pNP]b
μM
0
0.5
2
5
10
15
20
30
40
60
0
1.65
5.75
12.22
21.10
28.65
35.40
48.14
59.31
78.12
vpNPc
μM
min-1
3.059
2.343
1.931
1.636
1.465
1.343
1.169
1.043
0.864
vpNP/[CBH]d
min-1
2.59
2.52
2.41
2.26
2.15
2.06
1.89
1.76
1.57
[CBH]FAe
μM
1.18
0.93
0.80
0.72
0.68
0.65
0.62
0.59
0.55
[CBH]OAf
μM
[Glc]g
μM
1.32
1.57
1.70
1.78
1.82
1.85
1.88
1.91
1.95
0
20
65
128
209
274
331
434
525
688
vGlch
μM
min-1
kobs i
min-1
35.3
23.9
18.0
14.3
12.4
11.2
9.6
8.7
7.5
13.42
7.63
5.32
4.03
3.41
3.03
2.56
2.28
1.93
experiment was conducted in 50 mM sodium acetate pH 5.0 at 25 ºC with magnetic stirring 500 rpm.
Concentrations were following: [Avicel] = 10 g l-1, CBH was 2.5 μM TrCel7A, [pNPL] = 500 μM,
[BG] = 0.85 μM.
a
b
measured concentration of CBH released pNP. These figures were subjected to non-linear regression
according to the Equation (6) in order to get parameter values for velocity calculations.
c
velocity of pNP formation calculated according to the Equation (8) using parameter values from
previous step.
d
ratio vpNP/[CBH] was calculated from all-free line for TrCel7A according to the Equation (10) using
measured values for [pNP]b. These figures represents the expected values of (vpNP/[CBH]) at each
[pNP]b without the presence of cellulose.
e
concentration of CBH with free active site for pNPL hydrolysis is calculated from the figures in
previous two columns. [CBH]FA = vpNP/(vpNP/[CBH]) (Eq. 11).
f
concentration of CBH with occupied active site is the difference between total CBH ([CBH]Tot) and
[CBH]FA. In this example [CBH]OA = 2.5 μM - [CBH]FA.
g
measured concentration of CBH released glucose equivalents. These figures were subjected to nonlinear regression according to the Equation (7) in order to get parameter values for velocity
calculations.
h
velocity of glucose formation calculated according to the Equation (9) using parameter values from
previous step.
2
i
observed catalytic constant for cellobiose formation is now available from kobs = vGlc/2[CBH]OA (Eq.
2, velocity of cellobiose formation was considered to be equal to the half of the measured rate of
glucose formation in the presence of BG).
Table II Relative observed catalytic constants of TrCel7A, PcCel7D and their catalytic domains
(CD-s) on different celluloses
Relative kobs
Cellulose
Cel7A
Cel7D
Cel7A CD
Cel7D CD
100
46 ± 5
48 ± 4
44 ± 11
100
49 ± 6
117 ±26
60 ± 9
a
BMCC
Avicel
RAC
PWS
100
48 ± 5
57 ± 8
42 ± 8
%
100
43 ± 2
72 ± 5
41 ± 14
a
Relative kobs (as % of the value on BMCC) were first taken for each time point separately and table
lists the average values over all time points.
Table III Observed catalytic constants for catalytic domains (CD-s) compared to that of intact
enzymes
Observed catalytic constants for TrCel7A and its CD compared to corresponding figures for
PcCel7D
Relative kobs
Cellulose
Cel 7 ACD
Cel 7 A
Cel 7 DCD
Cel 7 D
%a
Cel 7 A
Cel 7 D
Cel 7 ACD
Cel 7 DCD
BMCC
Avicel
RAC
PWS
79 ±10b
76 ± 5
85 ± 8
81 ± 6
53 ± 7b
60 ± 7
83 ± 7
81 ± 12
56 ± 8c
62 ± 4
43 ± 3
58 ± 5
84 ±18d
78 ± 7
45 ± 11
58 ± 5
a
Relative kobs were first taken for each time point separately and table lists the average values over all
time points.
b
Relative kobs for CD as the % of the corresponding value for intact enzyme.
c
Relative kobs for TrCel7A as the % of the corresponding value for PcCel7D.
d
Relative kobs for TrCel7A CD as the % of the corresponding value for PcCel7D CD.
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