Experimental procedures-5

Enzyme

Catalytic of ABTS

• Mb variants were mixed with 7.5 mM of H₂O₂and 0.02 to 5 mM of ABTS.[Mb mutants] = 0.01 μM

• Monitored at 730 nm

• Michaelis-Menten plots and Linewaver-Burk plots

Scheme 5. Experimental procedures-5

2-2-12 Analysis of Peroxidase Activity

Activity for one-electron oxidation of ABTS was measured at room temperature in 100 mM potassium phosphate buffer (pH 7.0) on 8453 UV-Visible spectrophotometer. At least three experiments were performed for each experimental data point. The rate of the ABTS cation radical formation was monitored at 730 nm (ε730=1.4X10⁴M^-1 cm^-1) where the absorption of Mb was negligible. The reaction mixture contained ABTS (0.1-5 mM) and variable amounts of H2O2 (0.2-15 mM). The final concentration for Mb mutants was 0.01 μM.

3 Results and Discussion

3-1 All clones

The site directed mutagenesis strategy is a general method used in structural function studies for enzymes. In a previous study, MbH64D/V68L/I107M had been engineered to form an enzyme with peroxidase activity¹⁶. By using H2O2 and ABTS as substrates in a peroxidase activity assay, the activity of MbH64D/V68L/I107M was determined to be better than Mb^H64D/V68L. We further engineered the Leu-29 in MbH64D/V68L/I107M and His-93 in Mb^WT to construct MbL29X/H64D/V68L/I107M and Mb^H93X by site-saturated mutants. The X designation for each mutant represents the positions that were replaced with 19 amino acids. Inaddition, Val-17 was changed to Trp to construct Mb^V17W by site-directed mutagenesis. Phe-43 and Phe-46 were replaced with Tyr to construct Mb^F43Y and Mb^F46Y, respectively. The sequences of the above-mentioned clones were confirmed by DNA sequencing. The mutated plasmids were transformed into BL21(DE3) to facilitate protein expression. The results have been summarized in Table 8.

Mb Sequence Transform to BL21(DE3)

MbH64D/V68L/I107M sequence Transform to

BL21(DE3)

H93E(Glu) V V L29E(Glu) V V H93Q(Gln) V V L29Q(Gln) V V

H93G(Gly) V V L29G(Gly) V V

H93L(Lus) V V L29H(His) V V

H93I(Ile) V V L29I(Ile) V V

H93K(Lys) V V L29K(Lys) V V

H93M(Met) V V L29M(Met) V V

H93F(Phe) V V L29F(Phe) V V

H93P(Pro) V V L29P(Pro) V V

H93S(Ser) V V L29S(Ser) V V

H93T(Thr) V V L29T(Thr) V V

H93W(Trp) V V L29W(Trp) V V

H93Y(Tyr) V V L29Y(Tyr) V V

H93V(Val) V V L29V(Val) V V

Mb sequence Transform to BL21(DE3)

Mb sequence Transform to BL21(DE3) F43Y(Tyr) V V V17W(Trp) V V F46Y(Tyr) V V

Table 8. List of all clones.

3-2 Protein detection

We incubated all clones in 6 ml LB medium at 37 ℃ for 20 hours and then lysed the cells. To confirm that our clonescould express mutated Mbs efficiently in BL-21, the crude extracts were analyzed by Western blotting. The results revealed that all of our clones could express mutated Mbs efficiently (Fig. 16).

Lane 1: positive (Mb^WT) Lane 2: MbL29G/H64D/V68L/I107M Lane 3: MbL29A/H64D/V68L/I107M Lane 4: MbL29V/H64D/V68L/I107M Lane 5: MbL29I/H64D/V68L/I107M Lane 6: MbL29M/H64D/V68L/I107M Lane 7: MbL29S/H64D/V68L/I107M Lane 8: MbL29T/H64D/V68L/I107M Lane 9: MbL29C/H64D/V68L/I107M Lane 10: MbL29P/H64D/V68L/I107M

Lane 1: MbL29N/H64D/V68L/I107M Lane 2: MbL29Q/H64D/V68L/I107M Lane 3: MbL29F/H64D/V68L/I107M Lane 4: MbL29Y/H64D/V68L/I107M Lane 5: MbL29W/H64D/V68L/I107M Lane 6: MbL29K/H64D/V68L/I107M Lane 7: MbL29R/H64D/V68L/I107M Lane 8: MbL29H/H64D/V68L/I107M Lane 9: MbL29D/H64D/V68L/I107M Lane 10: MbL29E/H64D/V68L/I107M

Lane 1: positive (Mb^WT) Lane 2: Mb^H93G Lane 3: Mb^H93A Lane 4: Mb^H93V Lane 5: Mb^H93I Lane 6: Mb^H93M Lane 7: Mb^H93L Lane 8: Mb^H93S

Lane 9: Mb^H93T Lane 10: Mb^H93C

Lane 1: Mb^H93P Lane 2: Mb^H93N Lane 3: Mb^H93Q Lane 4: Mb^H93F Lane 5: Mb^H93Y Lane 6: Mb^H93W Lane 7: Mb^H93K Lane 8: Mb^H93R Lane 9: Mb^H93D Lane 10: Mb^H93E

Lane 1: positive (Mb^WT) Lane 2: Mb^V17W Lane 3: Mb^F43Y Lane 4: Mb^F46Y Figure 16. Western blotting experiments for clone expression

3-3 Activity Test of Crude Extract

In order to prove that the mutated Mb clones can express and confer peroxidase activity, we analyzed the peroxidase activity between BL-21 clones with MbH64D/V68L/I107M insert and BL-21 clones without insert. We detected the Soret band of Mb. The characteristic Soret band of Mb is expected to be around 410 nm and is contributed by heme in Mb. The Soret band will shift in accordance with different pH values, different solutions, or different mutants.

First, we lysed the cells to obtain, the crude extract. We then analyzed the difference in the UV-Vis spectrum. Acoording to the absorbance pattern at 410nm, the results indicated that the MbH64D/V68L/I107M clone could expressed the mutated protein consistently. No absorbance was detected at 410nm for the BL-21 clone without insert

(Fig. 17-a).

Next, we attempted to confirm functional peroxidase activity. The activity was from the MbH64D/V68L/I107M which was expressed by the inserted clone. 20 μl of extract was allowed to react with 1 ml mixed solution of 1 mM of ABTS and 5 mM of H2O2. The absorbance change detected at 730 nm, (ε730= 1.4 X10⁴ M^-1cm^-1) where the absorption of Mb was negligible, was recorded and compared. The results indicated;

there was no absorbance change at 730 nm for the crude extract of BL-21 clone without insert. On the other hand, the absorbance change at 730 nm for the crude extract of MbH64D/V68L/I107M clone was increased gradually. It was confirmed that the peroxidase activity was from the MbH64D/V68L/I107M, and not from other proteins in the BL-21 cell (Fig. 17-b).

240 340 440 540 640

Ab sorbance  (A U)

Wavelength (nm) UV‐Vis

Triple mutant BL‐21(no insert)

(b)

Figure 17. Comparisons of (a) wavelength scan and (b) peroxidase activity of crude extract from MbH64D/V68L/I107M clone (red) and BL-21 without insert (black)

Also, the influence of MbL29X/H64D/V68L/I107M, Mb^H93X, Mb^V17W, Mb^F43Y, and Mb^F46Y on peroxidase activity was analyzed using the crude extracts. The concentrations of all crude extracts were normalized by the absorbance of Soret band (shown in Appendix Figs. 27, 28, and 29). All of the crude extract concentrations were adjusted to 0.058 μM and mixed with 1 ml of 1 mM ABTS and 5 mM H2O2. The peroxidase activity of these crude proteins was analyzed spectrophotometrically at 25°C by using the H2O2-dependent oxidation of ABTS at 730 nm.Peroxidase activity results from of MbL29X/H64D/V68L/I107M, Mb^H93X, Mb^V17W, Mb^F43Y, and Mb^F46Y have been shown in Figure 18, by using the triple mutant MbH64D/V68L/I107M as positive control and wild type Mb as negative control. In crude extract of MbL29X/H64D/V68L/I107M, each clone exhibited peroxidase activity but worse than that of MbH64D/V68L/I107M (Fig. 18-a).

In crude extract of Mb^H93X, most did not have detectable peroxidase activity (Fig.

18-b). In crude extract of Mb^V17W, Mb^F43Y, and Mb^F46Y, only the Mb^V17W clone

showed activity (Fig. 18-c).

Figure 18. (a) The peroxidase activity of MbL29X/H64D/V68L/I107M clones. (b) The activity of H93X clones. (c) The peroxidase activity of V17W, F43Y, and F46Y

clones.

Wild type Triple mutant V17W F43Y F46Y

A(730nm)

Mb Variant

Peroxidase Activity of Crude Extract

Based on peroxidase activity results obtained using the crude cell extract, we further purified those clones with better activity. These were MbL29C/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, MbL29I/H64D/V68L/I107, MbL29M/H64D/V68L/I107M, and Mb^V17W.

3-4 Purification of mutated Mbs

To produce highly pure and large amount of Mb variants, we deferred to the research of Ribeiro et al.¹⁸, which produces inclusion bodies. When we isolated the inclusion bodies, we were to remove the miscellaneous proteins easily.

First, we used IPTG to induce Escheria coli. to produce inclusion bodies of our variant Mbs. Then, we lysed the cells. There were many inclusion bodies in the crude extracts. The inclusion bodies were isolated via centrifugation. The inclusion body was expected to be in the pellet because of its high density, and the miscellaneous proteins, which were in the supematent, were able to be efficiently removed. The inclusion bodies recovered were re-dissolved by Guanidine hydrochloride (Gdm-HCl).

Upon removal of the Gdm-HCl by dialysis, Mbs would slowly refold to avoid

subsequent aggregation. Last, Mbs were purified by using DEAE column, and analyzed by 20% SDS-PAGE to confirm the purity of the extracted proteins. The results showed that these Mb variants were pure and expressed in large quantities.

(Fig.19).

(a)

(b)

Figure 19. 20% SDS-PAGE analysis of purified mutated apoMb. (a) Lane M：

marker, lane 1：before adding IPTG, lane 2：after IPTG induced, and lane 3：

purified 17kDa Mb. (b) Lane M：marker, lane 1：Mb^WT, lane 2：Mb^H64D/V68L, lane 3：MbH64D/V68L/I107M, lane 4：Mb^V17W, lane 5：MbL29C/H64D/V68L/I107M, lane 6：

MbL29F/H64D/V68L/I107M, lane 7：MbL29I/H64D/V68L/I107M, and lane 8：

MbL29M/H64D/V68L/I107M

3-5 Reconstitution of mutated Mbs

In the extreme high or low pH range, Mbs are unfolding and the heme can exist as monomer or dimer. For this reason, Mb and heme were mixed in pH 12 solution and slowly adjusted to pH 6.8, in hopes of improving cofactor reconstitution. At the same time, heme was dissolved in pyridine to prevent aggregation, and dimethyl sulfoxide (DMSO) was added to the protein solution to increase the protein solution’s co-solubility. After the solution was completely adjusted to pH 6.8 and dialyzed, the heme that had not reconstituted with myoglobin was aggregated. We separated out that form of heme by passing through 0.45 μm filter and desalting column and discarded it.

To ensure the reconstituted Mbs had peroxidase activity, 10 μl of reconstituted MbH64D/V68L/I107M, apo-MbH64D/V68L/I107M, and heme only ([Mb]= 0.53 μg/ml=0.031 μM=[heme]) (Fig. 20) were reacted with ABTS (1 mM) and H2O2 (5 mM). Only the reconstituted protein had peroxidase activity (Fig. 21). This result was in agreement with the results published by Pfister et al.(Scheme 5).

Scheme 6. Heme dependence of tryptophan oxidation⁹

Figure 20. The UV-visible spectra of reconstituted MbH64D/V68LI107M and Apo-MbH64D/V68L/I107M in 100 mM potassium phosphate buffer at pH 7.0

0 0.1 0.2 0.3 0.4 0.5 0.6

350 400 450 500 550 600 650 700

Absorbance

Wavelength(nm)

Triple mutant & Apo-form

H64D/V68LI107M  Mb 

H64D/V68L/I107M  Apo‐Mb 

Figure 21. The absorbance change at 730 nm for MbH64D/V68LI107M, Apo-MbH64D/V68L/I107M, and heme in 100 mM potassium phosphate buffer at pH

7.0

3-6 Enzyme kinetics measurements

Enzyme kinetics is an older approach used to understand enzymatic mechanisms and remains to be the most important aspect. The purified MbL29C/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, MbL29I/H64D/V68L/I107, MbL29M/H64D/V68L/I107M, and Mb^V17W were subjected to enzyme kinetics assay. First, we analyzed the enzyme catalytic velocity of ABTS based on altering the concentration of ABTS. Mb variants were mixed with 7.5 mM of H2O2 and 0.01 to 5 mM of ABTS. The final concentration of Mb mutants was 0.01 μM. The results of the enzyme kinetics assay were analyzed by using Michaelis-Menten plots. The initial reaction rate v0 was used as Y axis and the substrate concentration [ABTS] was used as X axis. For enzymes that obey the Michaelis-Menten relationship, we used 1/ v0 as Y axis versus 1/ [ABTS] as X axis to draw the Linewaver-Burk plots. Also, we analyzed the enzyme catalytic velocity of H2O2 based on altering the concentration of H2O2. Next, Mb variants were mixed with 0.05 to 15 mM H2O2 and 1mM ABTS. The [H2O2]Michaelis-Menten and Linewaver-Burk plots were then analyzed. The Michaelis-Menten and

Linewaver-Burk plots of MbL29C/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, MbL29I/H64D/V68L/I107, MbL29M/H64D/V68L/I107M, MbH64D/V68L/I107M, Mb^H64D/V68L, and Mb^V17W have been shown in showed Appendix figure 30 and 31.

These two substrates are subject to substrate inhibition, especially H2O2. Alayash et al. had detailed the reactions of Mb with hydrogen peroxide in previous reports10,12,13,19. In their research, Mb reacted with H2O2 but not with ABTS. The ferryl iron species (compound I) changed to the ferric state via auto-reduction (Scheme 6). The mechanism of this process is not well understood, and the identity of the electron donor remains unknown. It may produce the compound III (superoxide radical anion), and it has been shown to slowly reduce to the ferric state. The peroxidase enzyme formed the compound III, which does not have peroxidase activity, when the concentration of H2O2 was high²⁰. In the experiment, the enzyme did not form the compound III when ABTS was added to the reaction.

Scheme 7. The rates of the slow kinetic processes were made based on a simple reaction scheme. ¹⁰

The steps of the peroxidase reaction are shown in equation 3 below. First, the enzyme is reacted with H2O2 to form compound I. The rate constant is represented by k1. Second, the ABTS reduced the complex to compound II. Finally, the ABTS continued to reduce the compound II until it reached the resting state and the iron of heme became ferric (Fig. 21). The constants for these reactions are represented by k2

and k3, respectively. Consequently, by changing the concentration of H2O2 we were able to measure the catalytic rate of H2O2.

Figure 22. General reaction cycles of heme proteins with peroxide⁹

(1)

(2)

(3) Equation 3. The equations of peroxidase catalysis

Mb^H64D/V68L, MbH64D/V68L/I107M, MbL29I/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, MbL29C/H64D/V68L/I107M, MbL29M/H64D/V68L/I107M, and Mb^V17W exhibited better peroxidase activity in crude extract analysis. We purified and reconstituted heme with these mutants; afterward, we finished the kinetic measurements. We have summarized the results in the table below and calculated the turnover number and catalytic efficiency.

ABTS

Mutated Mb apparent

V'max

L29I/H64D/V68L/I107M 0.052 0.211 5213 24737

L29F/H64D/V68L/I107M 0.026 0.153 2584 16913

L29C/H64D/V68L/I107M 0.017 0.102 1681 16542

L29M/H64D/V68L/I107M 0.057 0.196 5679 28954

V17W 0.010 0.063 994 15784

Table 9. Kinetic data of ABTS by mutated Mb

H²O²

Mutated Mb apparent

V'max

L29F/H64D/V68L/I107M   0.010 0.117 1008 8585

L29C/H64D/V68L/I107M  0.010 0.200 970 4840

L29M/H64D/V68L/I107M 0.071 1.039 7118 6853

V17W   0.008 0.168 765 4546

Table 10. Kinetic data of H2O2 by mutated Mb

0.00 

Figure 23. The catalytic efficiency of ABTS

0 

Figure 24. The catalytic efficiency of H2O2

Turnover number (also termed kcat) is defined as the maximum number of substrate molecules that an enzyme can convert to product per catalytic site per unit of time and can be calculated as follows: kcat = Vmax/[E]0. The catalytic efficiency allowed for comparison of peroxidase activities of the different mutants.

The activities of the quadruple mutants were worse than that of MbH64D/V68L/I107M. The result was determined by obtaining the kcat of ABTS and H2O2. The catalytic efficiency of H2O2 obtained for the quadruple mutants was a little worse than MbH64D/V68L/I107M. The catalytic efficiency of ABTS was the determining factor of activity. Every quadruple mutant was found to be worse than the triple mutant, which had an obvious difference when compared to other quadruple mutants. We supposed that the phenomenon was the steric effect of R group for amino residues.

The Leu-29 → Ile mutation decreased catalytic efficiency of ABTS by 1.4-fold.

This observation suggested that rotation of the substituted side chain about the Cα-Cβ bond was restricted due to branching at the β carbon and prevented ABTS access to the heme center. Such a direct steric interaction could explain the decrease in peroxidase activity by the replacement of Leu-29 with Phe. Yang et al. showed the same result in the Leu-68 → Ile¹¹. However, Hiner et al. and Brantley et al. found that the Leu-29 → Phe mutation decreased the rate of autooxidation 10-fold, but increased oxygen affinity by 15-fold^21,22. This data could explain why the MbL29F/H64D/V68L/I107M

catalytic efficiency of H2O2 appeared to be as good as Mb/H64D/V68L/I107M while the catalytic efficiency of ABTS was worse.

The Leu-29 → Met mutation decreased catalytic efficiency of ABTS by 1.2-fold.

This mutation caused a substantially different steric space as a result of Met replacement by Leu than that achieved when Leu is replaced by Ile. On the other hand, Met has a sulfur atom. Met stabilized the oxidation product of ABTS so that the Leu

→ Met made K'm higher than that of other mutants²³. The stable reactivity of ABTS

follows the order Cys >>> Trp > Tyr > His >>> Cys. The structure of Met is similar to that of Leu. However, Leu and Cys have very different steric spaces even if the Cys has the highest reactivity. The differences between the amino acids affects not only ABTS binding but also H2O2 binding.

Val-17 in helix A mediates steric interactions through Leu-69 to Val-68 in helix E.

The single mutant, Val-17 → Trp, effectively pushed the E helix closer to the heme iron through the Leu-69. Alayash et al. found that Mb^V68L had peroxidase activity¹⁰. Their data indicated that the rate constant of H2O2 of Mb^V68L is better than that of wild type by approximately 1.5 fold. The V17W, like Mb^V68L, had peroxidase activity, but it was worse than that of the double mutant, Mb^H64D/V68L.

Figure 25. The shortest distance of Leu-68 and Val-68 to iron atom by crystal of Mb^V68L(PDB: 1MLQ) and Mb^WT(PDB: 104M)

4 Conclusion and Future Perspective

The Leu-29 in Mb is located 7.64Å away from the iron center of heme. We supposed that this amino residue was a key point of subtractive protonation, for example ABTS. After replacing 19 amino acids with Leu-29 to construct MbL29X/H64D/V68L/I107M, we compared the peroxidase activity of MbL29X/H64D/V68L/I107M

with the peroxidase activity of MbH64D/V68L/I107M. The activity of H2O2 of the quadruple mutants was as good as the triple mutant, but the activity of ABTS was less than that of the triple mutant. The entrance of ABTS to the active site was seriously affected by the steric position of Leu-29. We plan to use a different kind of electron donor in future experiments, to create a different steric stabilization. This experiment will likely provide evidence that Leu-29 may particularly affect the activity of peroxidase.

The His-93 bound with heme could affect the position of iron. We, therefore, sought to determine whether the Mb peroxidase activity was due to the distal heme environment having been altered by amino acid mutation near the heme. In our research, there were no apparent differences between the 20 mutants of Mb^H93X. This result was quite similar to the results presented by Xiong et al²⁴. The protein backbone of Mb is known to fold and form a hydrophobic pocket that holds the heme in place. The mutated Mb peroxidase activity correlated with the environment around heme. The environment itself was dependent on the amino residue. For this reason, we believe the His-93 mutants did not support an environment for a functional peroxidase active site.

The single mutant, Val-17 → Trp, exhibited good catalytic efficiency. We found that V17W pushed the E helix close to the heme iron through Leu-69. It caused the

Val-68, which was on the E helix, to be nearer the heme iron. In the future, we can mutant the V17W with His-64 → Asp to test the peroixdase activity of Mb^V17W/H64D, and we can compare the peroxidase activity of Mb^H64D/V68L and Mb^V17W/H64D. In addition, we can compare the three-dimensional structure of Mb^H64D/V68L and Mb^V17W/H64D using an x-ray based approach. Ultimately, we may be able to prove whether the Mb^V68L and Mb^V17W exert similar effects.

The other single mutants, Phe-43→ Tyr and Phe-46 → Tyr, did not have obvious peroxidase activity. However, better activity was observed for Mb^F43Y and Mb^F46Y in comparison to wild type Mb. The two sites may not be the major site of peroxidase activity. We can mutate the F43Y and F46Y with the major site of peroxidase activity, like His-64 → Asp, and then compare the peroxidase activity of Mb^H64D. We anticipate that will aid in defining the influence of Phe-43 and Phe-46 on peroxidase activity.

Chroloperoxidase (CPO) is a heme-thiolate protein. The optimal pH value for its activity is 2 to 7. CPO halogenates organic substrates susceptible to electrophilic attack. It resembles cytochrome P450-dependent monooxygenases which also belong to the heme-thiolate protein family and can catalyze a multitude of biotechnologically important oxygen transfer reactions²⁵. Mb^H64D is a heme-imidazolate protein, but has the same distal heme environment in the activation of peroxide as CPO (Fig. 9 and 26). It is hoped that our understanding of the Mb mutation approach may also be applied to CPO to enhance its activity or to reduce its disadvantage (Table 11). In addition, CPO itself is resistant to acids and organic solvents. Therefore, our mutation approach may be useful for the examination and development of promising enzymes for industrial use.

(a)

(b)

  Figure 26. Stereoview of the CPO active site²⁶

CPO MB Residue

number Residue name Nearly atom

Distance

(Å) Possibly homologous residue of Mb

67 Val CG1 9.06

71 Ala CB 6.68

103 Phe CZ 5.96 F46

105 His NE2 ----

183 Glu OE2 5.06 H64D

186 Phe CE2 4.56 F43

Table 11. Possibly homologous residues of Mb and CPO

   

5 References

Barrick, D.; Wharton, D.; Champion, P. M. J. Phys. Chem. 2003, 107, 8156‐8165. 

  (25)  Hofrichter, M.; Ullrich, R. Appl Microbiol Biotechnol 2006, 71, 276‐88. 

  (26)  Sundaramoorthy, M.; Terner, J.; Poulos, T. L. Structure 1995, 3, 1367‐77. 

 

6 Appendix

400 420 440 460 480 500

Absorbance

400 420 440 460 480 500

Absorbance

‐0.1

400 420 440 460 480 500

Ab sorbance  

Figure 27. The Soret band of L29X/H64D/V68L/I107M clones

‐0.1

400 420 440 460 480 500

Ab sorbance  

0 0.05 0.1 0.15 0.2

350 450 550 650 750

Ab sorbance  

350 450 550 650 750

Ab sorbance  

0 0.05 0.1 0.15 0.2

350 450 550 650 750

Ab sorbance  

350 450 550 650 750

A

Figure 28. The Soret band of H93X clones

‐0.5

‐0.4

‐0.3

‐0.2

‐0.1 0 0.1 0.2 0.3 0.4 0.5

400 420 440 460 480 500

Ab sorbance  

WaveLength(nm)

UV‐Vis

Triple mutant V17W F43Y F46Y

Figure 29. The Soret band of V17W, F43Y, and F46Y clones

(a1)

(a2)

y = 3.9299x + 10.111 R² = 0.9987 0

50 100 150 200 250 300 350

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

^H64D/V68L

(b1)

(b2)

y = 2.7965x + 8.6511 R² = 0.9989 0

50 100 150 200 250 300

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

H64D/V68L/I107M

(c1)

(c2)

y = 3.455x + 17.641 R² = 0.999 0

50 100 150 200 250 300 350

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

L29M/H64D/V68L/I107M

(d1)

(d2)

y = 6.0502x + 59.337 R² = 0.9992 0

100 200 300 400 500 600

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

L29C/H64D/V68L/I107M

(e1)

(e2)

y = 5.8973x + 39.055 R² = 0.9983 0

100 200 300 400 500 600

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

L29F/H64D/V68L/I107M

(f1)

(f2)

y = 3.4149x + 23.158 R² = 0.9983 0

50 100 150 200 250 300 350

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

L29I/H64D/V68L/I107M

(g1)

(g2)

y = 6.3353x + 70.693 R² = 0.9974 0

100 200 300 400 500 600 700

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

^V17W

Figure 30. The Michaelies-Menten plot of mutant myoglobin catalyzing different concentration of ABTS, and the Lineweaver-Burk plot of mutant myoglobin for

calculating K’m and V’max. (a) Mb^H64D/V68L(b) MbH64D/V68L/I107M(c) MbL29M/H64D/V68L/I107M(d) MbL29C/H64D/V68L/I107M (e) MbL29F/H64D/V68L/I107M (f)

MbL29I/H64D/V68L/I107M (g) Mb^V17W

(a1)

(a2)

y = 15.279x + 8.5921 R² = 0.9982

0 50 100 150 200 250 300 350

‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

^H64D/V68L

(b1)

(b2)

y = 12.983x + 6.7978 R² = 0.9994 0

50 100 150 200 250 300

‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

H64D/V68L/I107M

(c1)

(c2)

y = 14.592x + 14.055 R² = 0.9979 0

50 100 150 200 250 300 350

‐10 ‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

L29M/H64D/V68L/I107M

(d1)

(d2)

y = 20.629x + 103.55 R² = 0.9976 0

100 200 300 400 500 600

‐10 ‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

L29C/H64D/V68L/I107M

(e1)

(e2)

y = 11.781x + 97.067 R² = 0.9977 0

50 100 150 200 250 300 350 400

‐10 ‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

L29F/H64D/V68L/I107M

(f1)

(f2)

y = 4.0365x + 19.525 R² = 0.9977 0

50 100 150 200 250 300 350 400

‐20 0 20 40 60 80 100

1/V(min/mM)

1/[S](1/mM)

Mb

L29I/H64D/V68L/I107M

(g1)

(g2)

y = 21.697x + 134.94 R² = 0.9977 0

100 200 300 400 500 600

‐10 ‐5 0 5 10 15 20 25

1/V(min/mM)

1/[S](1/mM)

Mb

^V17W

Figure 31. The Michaelies-Menten plot of mutant myoglobin catalyzing different concentration of H2O2, and the Lineweaver-Burk plot of mutant myoglobin for

calculating K’m and V’max. (a) Mb^H64D/V68L(b) MbH64D/V68L/I107M(c) MbL29M/H64D/V68L/I107M(d) MbL29C/H64D/V68L/I107M (e) MbL29F/H64D/V68L/I107M (f)

MbL29I/H64D/V68L/I107M (g) Mb^V17W

在文檔中 Leu29與His93位置之飽和定點突變抹香鯨肌紅蛋白對其過氧化能力之影響 (頁 48-0)