A. Construction of site-directed mutagenesis of RM-CK and M1-CK mutants
A rabbit muscle cDNA library was purchased from Merck, and RM-CK was cloned with primer pairs 5′-atcccatatgccgttcggcaac-3′ and 5′- aaaactcgagctacttctgggc-3′. The PCR product was digested with NdeI and XhoI, and then ligated into pET 28a (Wu et al., 2011). M1-CK was constructed previously (Sun et al., 1998), it is also digested with NdeI and XhoI, and then ligated into pET28a.
To mutate glycine to asparagine, aspartic acid, lysine or leucine at residue 268 of RM-CK, bridge PCR method was used in site directed mutagenesis on RM-CK-pET28a clone. The primers were displayed in Table 2.
Similarly, mutated asparagines to glycine, aspartic acid, lysine or leucine at residue 268 of M1-CK, bridge PCR method was also used in site directed mutagenesis on M1-CK-pET28a clone, the primers were displayed in Table 2.
5 μl Taq DNA polymerase 10x buffer, 4 μl dNTP, 32.5μl d.d.H2O , 1 μl primer (100 μM) twice, 1 μl template (RM-CK-pET28a or M1-CK-pET28a), 5 μl MgCl2 (25 mM) and 0.5 μl Taq DNA polymerase are used in the first PCR. The PCR program was one cycle of 94 oC for 5 min, 35 cycles of denature 94 oC for 20 sec, annealing 50 oC for 20 sec and extension 70 oC for 90 sec, and final extension of one cycle at 70 oC for 5 min.
25
The first PCR products were separated by 1.5 % DNA agarose gel and purified by QIAquick Gel Extraction Kit (QIAGEN). For second PCR, 5 μl the purified DNA fragment twice, 5 μl Pfu DNA polymerase 10x buffer, 4 μl dNTP, 25 μl d.d.H2O, 5 μl MgCl2 (25 mM) and 1 μl Pfu DNA polymerase were added. The PCR program was one cycle of 94 oC for 5 min, 15 cycles of denature 94 oC for 20 sec, annealing 52 oC for 20 sec and extension 70 oC for 90 sec, and final extension of one cycle at 70 oC for 5 min.
When second PCR finished, 2 μl forward and reverse primers were added directly for the third PCR. The PCR program was one cycle of 94 oC for 5 min, 25 cycles of denature 94 oC for 20 sec, annealing 52 oC for 20 sec and extension 70 oC for 90 sec, and final extension of one cycle at 70 oC for 5 min. The third PCR products were purified by QIAquick PCR Purification kit (QIAGEN).
Subsequently, NdeI and XhoI were used to double digest the third PCR purification products and pET28a vector. 4 μl d.d.H2O, 2 μl 10x Fastdigest buffer, 10 μl third PCR purification products or pET28a vector, 2 μl FastDigest NdeI and 2 μl FastDigest XhoI were added to the 1.5 ml microcentrifuge tube, then incubated at 37 oC for 20 minutes for digestion. These digested products were subjected to 1 % DNA agarose gel electrophoresis then used QIAquick Gel Extraction Kit (QIAGEN) to purify the sticky-end DNA fragment and pET28a. 3.9 μl sticky-end DNA fragment, 8 μl sticky-end pET28a, 1.5 μl 10x T4 DNA ligase buffer, 1 μl T4 DNA ligase and 0.6 μl ATP (25 mM )
26
are used in ligation at 4 oC overnight.
2 μl ligation product was added to the 100 μl ECOSTM 101 competent cells (storage at -80 oC, Yeastern Biotech, Taipei). Placed on ice for 30 minutes, heat-shocked for 45 seconds at 42 oC water bath then replaced on ice for 2 minutes. 1 ml SOC medium was added to the tubes containing transformed cells, and then incubated at 37 oC for 1 hour with shaking. The transformation cultures were plated on LB/kanamycin plates (LB Agar/Kanamycin 50, RH: 60% ± 15, CMP) and incubated at 37 oC overnight. A single colony was picked and cultured in LB medium with kanamycin overnight (LB : 10 mg/ml kanamycin = 1000:1). Mini-plasmid extraction kit (VIOGENE) was used to purify the recombinant plasmid DNA, then the size of plasmid was confirmed by 1 % DNA agarose gel electrophoresis. After confirming the sequences by commercial company, the recombinant plasmid DNAs were stored at -20 oC.
B. Production of protein of RM-CK and M1-CK mutants
2μl recombinant plasmid DNA was added to 100 μl ECOSTM21 competent cells (storage at -80 oC, Yeastern Biotech, Taipei). Placed on ice for 30 minutes, heat-shocked for 45 seconds at 42 oC water bath then replaced on ice for 2 minutes. 1 ml SOC medium was added to the tubes containing transformed cells, and then incubated at 37
oC for 1 hour with shaking. The transformation cultures were plate on LB/kanamycin
27
plates (LB Agar/Kanamycin 50, RH: 60% ± 15, CMP) and incubated at 37 oC overnight.
A single colony was picked and cultured in 100 ml LB medium with 100 μl kanamycin (10 mg/ml) at 37 oC overnight. Next day, it was added to 900 ml LB with 1000 μl kanamycin (10 mg/ml), incubated at 37 oC for 4 hour with shaking, and then 1000 μl IPTG (0.4M) was added, incubated at 37 oC for 1.5 hour with shaking. The culture medium was centrifuged for 30 minutes, supernatant discarded and pellet resuspended in 20 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). After sonication (Time: 5 minutes, Pulse: 10, 05, Amp: 1, 30%, SONICS) and centrifuged at 30 minutes at 4 oC, supernatant was filtrated using a 0.2 μm filter (Sartolab B/T, 150, 0.2 PES, R/FST, Sartorius, made in USA) and applied in to a nickel column.
The nickel column was prepared previous day. The histag binding agarose was purchased from Bioman (Cat No: PBP001.100), and filled with 1x charge buffer overnight. Before protein purification, charge buffer was replaced by 1x binding buffer.
The target protein was eluted with elution buffer (0.2 M imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9), and it was eluted between 200 mM to 300 mM imidazole. The experiment was conducted at 4 oC.
Elution product was dialysed in 30 mM Tris, 10 mM MgCl2, 1 mM DTT, pH 7.1 at 4
28
oC for at least 36 hours. The dialysis membrane was purchased from Spectrum
Laboratories, Inc. (MWCO:6-8000, reorder no:132 660). After dialysis, the protein was concentrated with an Amicon Ultra 15 filtration tube (MWCO: 10000, Millipore Corp., Billerica, MA, USA). The concentrated protein solution was mixed with protein storage buffer in the ratio of 2/3 (v/v), and storage at -30 oC. The final protein storage condition was 30 mM Tris, 10 mM MgCl2, 1 mM DTT and 50% glycerol. Protein concentration was estimated by Coomassie plus protein assay reagent (Thermo, standard source:
bovine serum albumin, concentration: 2.0 mg/ml in a 0.9 % aqueous). The spectrophotometric absorption was measured at 595 nm (Synergy HT ELISA Reader, BIO-TEK).
C. Specific activity assay
M-CK specific activity assay was based on the method described by Hughes and modified to fit different assay conditions (Hughes, 1962). Since the Sigma M-CK activity assay kit (SIGMA Diagnostic, No. 520; Sigma-Aldrich. St Louis, MO, USA), which was used in our previous studies, was no longer available. Therefore, we prepared all chemicals and all stock solution as mention in the Sigma M-CK activity assay kit.
The basal reaction buffer of M-CK activity assay was 30 mM Tris, 10 mM MgCl2,
29
and 1 mM DTT (1,4-Dithio-DL-threitol). Assay buffer pHs were adjusted to 7.1, 7.4, 7.7 or 8.0 at either 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, 10 °C or 5 °C. The pH of the assay buffers were adjusted by titration with HCl, at the assay temperatures.
25 μl 3x activity assay buffer (90 mM Tris, 30 mM MgCl2, and 3 mM DTT), 25 μl 40mM PCr and 25 μl 3.2 mM ADP were added to 1.5 ml microcentrifuge tube (Axygen, MCT-175-C). After preincubated 15 minutes in water bath, 5 μl enzyme (2.5 μg/ml, dilute with 1x activity assay buffer) was added to the tube, but blank was added 5 μl 1x activity assay buffer. The final reaction condition was 12.5 mM PCr, 1 mM ADP and 0.0125 ng enzyme in the reaction solution of 80 μl. After 15 minutes, the reaction was terminated with 20 μl stop solution (50 Mm p-hydroxyl-mercuribenzoate), then 100 μl α-naphthol (2%, w/v) and 100 μl diacetyl (1:200, v/v) were added for the colorimetric reaction. Finally, 700 μl d.d.H2O was added and mixed, then incubated at 37°C for 15 minutes. Centrifugation for 5 minutes, 300 μl supernatant added to 96 microwell (Thermo, nuncTM) and spectrophotometric absorption was measured at 520 nm (Synergy HT ELISA Reader, BIO-TEK). Creatine produced in each reaction was quantified using a creatine standard curve (0.08, 0.064, 0.048, 0.032, 0.016, 0, μ mole) and specific activity U was defined as 1 μmole of creatine formed per min per mg of enzyme. All the reactions were repeated at least 3 times and with more than 1 batch of recombinant proteins.
30
D. Kinetic assay and data analysis
Kinetic analyses of CKs were carried out as described in Cleland (1979) (Cleland, 1979). The enzyme specific activity assays for biochemical kinetic analysis of CKs were carried out only at pH 7.1 and 8.0, and at either 35 °C, 25 °C, 15 °C or 5 °C. ADP concentration range was from 0.1 to 2 mM and PCr was at 120 mM when determining the KmADP. PCr concentration range was from 0.5 to 120 mM and ADP was at 2 mM when determining the KmPCr. In each reaction, 6 ng of enzyme was added, and the reaction time was 7 minutes for the 35 °C reaction, 15 minutes for the 25 °C and 15 °C reactions and 45 minutes for the 5°C with volume of 40 μl each. Kcat, KmADP and KmPCr
were calculated using the double reciprocal plot of Michaelis–Menten equation (Marangoni, 2003).
v = Vmax (S) (Km + (S) )-1,
where v was reaction rate obtained from Cr formation rate, (S) was the initial
concentration of one of the substrates, while the other substrate concentration was fixed, Vmax is defined as the maximum reaction velocity.
Vmax = kcat (Etotal),
where Etotal = total enzyme concentration.
In the steady-state case, v-1=Km(S)-1Kcat-1 (Etotal)-1+kcat-1 (Etotal)-1, and the graph v-1v.
(S)-1 was plotted.
31
On the graph, when (S)-1 = 0, v-1 = kcat (Etotal)-1, and when v-1 = 0, (S)-1 = -Km-1
All the reactions were repeated at least 3 times and with more than 1 batch of recombinant proteins. Molecular weights were calculated using the ExPASy Proteomics Server website (Swiss Institute of Bioinformatics), and concentrations of enzymes were calculated using these data.
E. Thermal stability assay
Thermal stability experiments were carried out in the activity assay buffer (30 mM Tris, 10 mM MgCl2, and 1 mM DTT) at pH 7.1 or 8.0. Enzyme was diluted with 1x activity assay buffer to 2.5 μg/ml, and then preincubated at 10, 20, 30, 40, 50, 60 or 70°
C for 30 min, then, cooled on ice.
25 μl 3x activity assay buffer (90 mM Tris, 30 mM MgCl2, and 3 mM DTT), 25μl 40mM PCr and 25μl 3.2 mM ADP were added to 1.5 ml microcentrifuge tube (Axygen, MCT-175-C). After preincubated 15 minutes at 35°C at water bath, 5 μl enzyme was added to the tube, but blank was added 5 μl 1x activity assay buffer. The final reaction condition was 12.5 mM PCr, 1 mM ADP and 0.0125 ng enzyme in the reaction solution of 80 μl. After 15 minutes reaction at 35° C, the reaction was terminated with 20 μl stop solution (50 Mm p-hydroxyl-mercuribenzoate), then 100 μl α-naphthol (2%, w/v) and
32
100 μl diacetyl (1:200, v/v) were added for the colorimetric reaction. Finally, 700μl d.d.H2O was added and well mixed, then incubated at 37°C for 15 minutes.
Centrifugation for 5 minutes, 300 μl supernatant added to 96 microwell (Thermo, nuncTM) and spectrophotometric absorption was measured at 520 nm (Synergy HT ELISA Reader, BIO-TEK). Creatine produced in each reaction was quantified using a creatine standard curve (0.08, 0.064, 0.048, 0.032, 0.016, 0, μ mole)
At least three repeats were carried out, and the highest specific activity was taken as 100% (Zhao et al., 2006a). The specific activities of all the M-CKs in each condition were assayed from three different batches of recombinant protein preparation and each preparation assayed for more than three times.
F. Circular dichroism
Circular dichroism (CD) spectra were recorded on a Jasco J715 spectropolarimeter (Jasco International, Tokyo, Japan) at temperatures 35 °C, 25 °C, 15 °C or 5 °C (Kelly et al., 2005). For far-UV spectrum, a 1 mm path length quartz cuvette (Hellma Analytics, order number: 165-1-40, type: 165-QS) was used at a scan speed of 20 nm.min−1 from 200 nm to 250 nm. The solution for CD contained 2.32 μM of M-CK protein in activity assay buffer at pH 7.1 or 8.0, in the absence of ADP and PCr. Data were collected per 0.1 nm and an average of 8 spectra were corrected by subtraction of
33
spectra recorded on the activity assay buffer in the absence of enzyme.
G. Structure modeling
Since the crystal structure of RM-CK and RM-CK G268N had been resolved (Rao et al., 1998; Wu et al., 2011), and the primary sequences of RM-CK and M1-CK share 86% identity, therefore, we could use RM-CK as template to modeling the 3D structure of RM-CK mutants, M1-CK and M1-CK mutants.
Since M-CK is highly conserved enzyme, the homology modeling methods was used to model the structure of our target proteins (Marti-Renom et al., 2000), and the MODELLER is the free program to carry out homology modeling for model building (Eswar et al., 2007; http://salilab.org/modeller/). The template for build structure was the PDB 2CRK (Rao et al., 1998). The output 3D structures were displayed by the free program included PyMol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, San Carlos, CA, USA; http://www.pymol.org.), Swiss-PdbViewer (Guex and Peitsch, 1997; http://www.expasy.org/spdbv/). The solvent accessible surface of each residue was calculated by free program MolMol (Koradi et al., 1996), and the Cα distances were calculated by Swiss-PdbViewer.
34
Results
A. Construction of site-directed mutagenesis of RM-CK and M1-CK mutants
Using RM-CK-pET28a or M1-CK-pET28a as template, the 268 residue mutants were constructed by overlap-extension technique. The PCR product and pET28a were double digestion with NdeI and XhoI. After purified by gel extraction, the DNA fragment was ligated to the pET28a vector (Fig. 1). These resulting plasmids were designated as RM-CK G268N, RM-CK G268D, RM-CK G268K, RM-CK G268L, M1-CK N268G, M1-CK N268D, M1-CK N268K, M1-CK N268L. The inserted DNA fragment was 1146 base pairs; pET28a was 5369 base pairs and the total size of recombinant plasmid was 6834 base pairs(Fig 1). The plasmids were storage at -20 oC.
B. Production of protein of RM-CK and M1-CK mutants
The recombinant plasmids were transformed in to ECOSTM21 competent cells to express the recombinant protein. The protein were applied in to a nickel column and eluted with elution buffer (0.2 M imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). The recombinant protein appeared at 200 mM and 300 mM imidazole (Fig. 2). All the mutant proteins are the same size as the wild-type protein, about 45 kDa (Fig. 3). After
35
dialysis in 30 mM Tris, 10 mM MgCl2, 1 mM DTT, pH 7.1 at 4 oC, the proteins were concentrated with Amicon Ultra 15 filtration tubes. The concentrated protein solution was mixed with protein storage buffer and storage at -30 oC. The final protein storage condition was 30 mM Tris, 10 mM MgCl2, 1 mM DTT and 50% glycerol.
C. Specific activity assay
Polar side chain of residue 268 of muscle form creatine kinase show higher activity
at low temperature
The residue 268 of M1-CK and RM-CK were mutated to glycine, asparagine, aspartic acid, lysine or leucine, individually. M-CK specific activity assay was based on the method described by Hughes and modified to fit different assay conditions (Hughes, 1962). The specific activities were assayed at pHs 7.1, 7.4, 7.7 or 8.0 at either 40 °C, 35
°C, 30 °C, 25 °C, 20 °C, 15 °C, 10 °C or 5 °C.
The four M1-CK mutants showed different pH-temperature specific activity patterns compared to wild-type M1-CK (Fig 4). M1-CK, M1-CK N268D, M1-CK N268G, M1-CK N268K and M1-CK N268L showed their highest specific activity of 445 ± 42.44 U, 446 ± 40.75 U, 385 ± 41.48 U, 364 ± 22.27 and U 305± 32.2 (mean ± S.D.), respectively, at pH 7.1 and 30 °C (Table 4).
At pH 7.1 and 7.4, M1-CK had highest activity at 30 °C, and started to decrease at
36
temperature > 30 and <30 °C, but still remain >100 U activity at 15 and 10 °C. At pH 7.7, M1-CK had highest activity of 151 ± 4.82 at 30 °C and 143 ± 7.4 U at 15 °C, and still remain 95.5± 5.54 U at 10 °C. At pH 8.0, M1-CK was able to maintain its activity around 70 U at 15 and 10 °C. Activity trend of M1-CK N268G was similar to M1-CK at pHs 7.1, 7.4 and 7.7, but lower at all temperature. At pH 8.0, compared to M1-CK, M1-CK N268G was 22% and 34% less active at 15 and 10 °C, respectively. M1-CK N268D was the mutant enzyme of highest activity at 35 and 30°C at all pHs, and maintained activity of 127± 3.2 and 90.6 ± 3.87 at 15 and 10 °C, respectively at pH 7.7.
At pH 8.0, compared to M1-CK, M1-CK N268D was 11.5 % and 13 % less active at 15 and 10 °C, respectively. The activity of M1-CK N268K was lower than M1-CK N268D and almost the same as M1-CK N268G at 15 °C at either pH 7.7 or 8.0 and at pH 8.0, at 10 °C. At pH 8.0, at 10 °C, activity of M1-CK N268K was lower than M1-CK N268D and higher than M1-CK N268G, and it was 24% less activity compared to M1-CK.
M1-CK N268K and M1-CK N268D could recover activities at 15 °C of all pHs and at 10 °C, pH 8.0. M1-CK N268L showed the same activity pattern as M1-CK N268G at low temperature. M1-CK N268L showed the lowest activity and was 62.8 %, 39.7 %, 55 % and 67 % less activity compared to M1-CK at 15 and 10 °C at either pH 7.7 or 8.0, respectively.
The four RM-CK mutants also showed different pH-temperature specific activity
37
patterns compared to the wild-type (Fig 5). RM-CK, RM-CK G268N, RM-CK G268K, RM-CK G268D, and RM-CK G268L showed their highest specific activity of 352.76 ± 24 U, 436.5 ± 7.1 U, 407.6 ± 1.1 U, 401.2 ± 3.7 U, and 314.5 ± 4.6 U (mean ± S.D.), respectively, at pH 7.1 at 35 °C (Table 5).
RM-CK had higher activity around 35 to 25°C and decreases as temperature decreased. At pH 8.0 at 10°C, RM-CK lost 92% of its activity (29 ± 3.1 U). RM-CK G268N showed the similar activity pattern as RM-CK at high temperature (40 to 25 °C), but had higher activity than RM-CK at low temperature (15 and 10°C) at pHs 7.7 and 8.0. At pH 7.7, RM-CK G268N showed 50% higher activity compared to RM-CK at 10°C. RM-CK G268N, as M1-CK, maintained its activity of 66.2± 7.6 U and showed 56% higher activity compared to RM-CK at pH 8.0, at 10°C. RM-CK G268D and RM-CK G268K had higher activities than RM-CK and RM-CK G268L at all pHs and temperatures, and their activities were similar to RM-CK G268N excepted at 10°C, at pHs 7.7 and 8.0. The activity of RM-CK G268L was the same as RM-CK at pH 7.7, at low temperature (15 to 5°C). At pH 8.0 at 10°C, RM-CK G268L showed lowest activity than other.
A polar side chain of residue 268 of muscle-form creatine kinase showed higher activity than non-polar residue at low temperature (15 and 10 °C), at high pHs (7.7 and 8.0).
38
D. Kinetic assay
Polar side chain of residue 268 could maintain stable Km and Kcat or increased Kcat..
The kinetic parameters, KmADP, KmPCr, KcatADP, KcatPCr, of each enzyme were assay at 35, 25, 15 and 5°C at either pH 7.1 or 8.0.
The KmADP trend of M1-CK N268G resembled that of M1-CK N268K at pH 7.1, but lowers all temperature (Fig 6). At pH 7.1, M1-CK, M1-CK N268D and M1-CK N268L had similar KmADP pattern. At pH 8.0, M1-CK N268G, M1-CK N268K and M1-CK N268L showed similar KmADP pattern and M1-CK N268L was the lowest. M1-CK kept its KmADP at different temperatures but rose slightly at 5°C at pH 8.0. M1-CK N268D had similar KmADP to others except at 15°C, at pH 8.0.
At pH 7.1, the KmADP of RM-CK and its mutants were not significantly different at all temperatures expect RM-CK G268D was lower than RM-CK and other mutants at 35°C (Fig 8). At pH 8.0, RM-CK G268K resembled that of RM-CK G268L and higher at all temperatures, both of these two mutants maintained their KmADP at 35, 25 and 15°C but decreased at 5°C. The KmADP of RM-CK and RM-CK G268D decreased at 25°C then maintained it as temperature decreased. RM-CK G268N maintained its KmADP at pH 8.0 at all temperatures.
At pH 7.1, the KmPCr of M1-CK and its mutants increased as temperature decreased (Fig 6). At pH 7.1, from 25 to 5°C, M1-CK N268G had highest KmPCr and M1-CK had
39
lowest KmPCr. M1-CK N268D, M1-CK N268K and M1-CK N268L were not significantly different at pH 7.1 from 25 to 5°C. At pH 8.0, M1-CK N268G showed no significantly difference with M1-CK N268D at all temperatures, and M1-CK N268L was higher than them at 25 and 15°C. At pH 8.0, M1-CK was slightly decreased as temperature decreased, but generally speaking, maintained its KmPCr at all temperatures.
M1-CK N268K showed highest KmPCr at 35 and 25°C but decreased at 15 °C at pH 8.0.
At pH 7.1, the KmPCr of RM-CK and its mutants increased as temperature decreased except RM-CK G268N (Fig 8). At pH 7.1, RM-CK G268N maintained its KmPCr at 15 and 5°C. At pH 8.0, the KmPCr of RM-CK and RM-CK G268N decreased slightly as temperature decreased, on the other hand, those of RM-CK G268K and RM-CK G268L also decreased slightly as temperature decreased but increased at 5°C. At pH 8.0, KmPCr
of RM-CK G268D decreased more significantly than other mutants when temperature decreased. KmPCr of RM-CK and mutants were no significantly different at 15°C, pH 8.0.
At pH 7.1, the KcatADP of M1-CK and mutants decreased as temperature decreased, but, unlike other four mutants decreased sharply, M1-CK decreased gently (Fig 7). At pH 8.0, M1-CK could maintain its KcatADP when temperature changed; KcatADP of M1-CK N268G decreased slightly as temperature decreased. The KcatADP of M1-CK N268D, M1-CK N268K and M1-CK N268L undulated at pH 8.0.
40
At pH 7.1, KcatADP of RM-CK and mutants decreased as temperature decreased, and there was no significantly difference at 25, 15 and 5°C (Fig 9). At pH 8.0, KcatADP of RM-CK and mutants also decreased as temperature decreased, and there was not significantly difference at 35 and 5°C. At pH 8.0, at 25 and 15 °C, they also showed the same KcatADP except that of RM-CK G268K was higher than other.
The KcatPCr of M1-CK and mutants decreased as temperature decreased, at pH 7.1, and compared to other mutants, M1-CK decreased more gently (Fig 7). The KcatPCr of M1-CK N268D resembled that of M1-CK N268K at pH 7.1. At pH 8.0, M1-CK could maintain its KcatPCr as temperature decreased; M1-CK N268G also could maintain its KcatPCr at 35, 25 and 15°C, but decreased at 5°C. At pH 8.0, KcatPCr of M1-CK N268D, M1-CK N268K and M1-CK N268L were decreased as temperature decreased, but at 25°C, KcatPCr of M1-CK N268K and M1-CK N268L increased significantly.
The KcatPCr of RM-CK and mutants decreased as temperature decreased, at pH 7.1,
The KcatPCr of RM-CK and mutants decreased as temperature decreased, at pH 7.1,