Biology Assignment

Indian Journal of Biochemistry & Biophysics Vol. 50, February 2013, pp. 64-71

Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP

Gouri Chaudhuri 1 , Saswata Chatterjee

1 , P Venu-Babu

2 , K Ramasamy

3 and W Richard Thilagaraj

1*

1 Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, Tamil Nadu, India

2 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

3 Tamil Nadu Agricultural University,

Coimbatore 641003, Tamil Nadu, India

Received 21 June 2012; revised 03 December 2012

The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as Vmax, Km and Kcat under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP

in 50 mM

Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37°C: Vmax, 3.12 and 1.6 µmoles min

-1 unit

-1 ; Km, 7.6 × 10

-4 M and 4 × 10

-4 M; and Kcat, 82.98 s

-1 and 42.55 s

-1 , respectively. CIAP displayed a high

temperature optimum of 45°C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu

2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained.

Pi generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.

Keywords: Calf intestinal alkaline phosphatase, Glycine-NaOH buffer, Hydrolysis, p-Nitrophenyl phosphate, Temperature, Tris-HCl buffer, Bioremediation.

Several pollutants are discharged everyday into air, soil and water from a large number of industries, which is of global concern. The bioremediation technology is cost-effective, eco-friendly method compared to other traditional physico-chemical methods, such as precipitation, coagulation, electro- chemical reduction, ion-exchange etc. There are different methods to use microorganisms for the purpose of bioremediation. But, the disadvantage of using microorganisms is that for their growth, optimum conditions are required to be maintained

1 .

On the other hand, ‘Green chemistry’ i.e., use of biocatalysts for remediation of pollutants has opened a new area of research globally

2 . In the past few

years, enzymatic bioremediation (known as ‘White Biotechnology’) has become an effective alternative to remediate heavy-metals from waste

3 . Alkaline

phosphatases (AP or ALP, EC 3.1.3.1.) are ubiquitous enzymes found in most species from prokaryotes to eukaryotes

4 . They can hydrolyze a wide range

of monophosphate ester substrates and exhibit phosphotransferase and protein phosphatase activities

5 .

There has been considerable effort in recent years towards the application of ALPs for bioremediation of heavy metals and radionuclides from nuclear wastes. Thus, it is required to study the kinetic behaviour of ALP. The ALP from E. coli has been the focus of several studies dealing with molecular properties, subunit composition and catalytic mechanism, however, studies with mammalian ALPs, especially calf intestinal alkaline phosphatase (CIAP) are lacking. Mammalian ALPs are glycoproteins and exist as different isoenzymes such as placental (PLAP), germ cell (GCAP), intestinal (CIAP) and tissue non-specific isoforms

4 .

Mammalian ALP is a very important enzyme physiologically and is an important component of medical diagnosis

6 . Like the E. coli isoforms, the

mammalian ALPs are also zinc-metalloenzymes that can be activated by Mg

2+ and Zn

2+ , both ions being

—————— * Corresponding author:

Tel.: 91-9840712683; Fax: 91-044-27453903 E-mail: [email protected] Abbreviations: AP or ALP, alkaline phosphatase; CIAP, calf intestinal alkaline phosphatase; GCAP, germ cell alkaline phosphatase; pNP, p-nitrophenol; pNPP, p-nitrophenyl phosphate; PLAP, placental alkaline phosphatase.

CHAUDHURI et al: KINETIC BEHAVIOUR OF CALF INTESTINAL ALP WITH pNPP

65

essential for the catalytic activity and structural stability of ALP. Mammalian ALPs show 25-35% sequence identity with the E. coli enzyme in those regions of the protein that assume α-helix and β-strand secondary structures and also those regions that are critical for catalytic activity

7 . The catalytic

residues i.e., Asp91, Ser92 and Arg166 and ligands coordinating the divalent metal ions (Zn

2+ and Mg

2+ )

are all conserved 8 . These structural similarities

suggest that mammalian ALPs may catalyze hydrolysis of both pyrophosphate and orthophosphate moieties via a mechanism similar to that of E. coli enzyme

9 .

The ability of purified human liver and small intestinal ALPs to act on a number of organic di- and tri-phosphates of physiological significance is reported previously

10 . The ALP from calf intestinal

mucosa (CIAP) is one of the most active ALPs known. Although the kinetic behaviour of CIAP with 4-methylumbelliferyl phosphate is reported

11 , in the

present paper, we report the kinetic behaviour of CIAP with p-nitrophenyl phosphate (pNPP) and the effects of pH, buffer, substrate concentration, temperature and incubation time on the hydrolysis of pNPP by the enzyme, in order to understand the efficiency of CIAP for enhancing bioprecipitation of heavy metals. Further, we also demonstrate precipitation of copper by CIAP-mediated catalysis of pNPP. Materials and Methods

Two buffers viz., 50 mM Tris-HCl and 100 mM glycine-NaOH were used during the course of experiments in the range of pH 8 to 12. A purified ALP from calf intestinal mucosa (Invitrogen) was used all throughout. One unit of enzyme hydrolyzed 1 µmole of pNPP in 1 min at 37°C. The enzyme was stored at -20°C till use. pNPP (Sigma) was used as substrate for the CIAP. The substrate as well as the working stock was stored at 4°C. Determination of CIAP-catalyzed hydrolysis of pNPP

The catalytic activity was followed by monitoring the increase in the absorbance at 405 nm from p-nitrophenol (pNP) generated in an enzyme- catalyzed hydrolysis of pNPP using Shimadzu UV spectrophotometer. All hydrolytic reactions for studying the effects of different substrate concentrations (0.2, 0.5, 1, 1.5, 2, 2.5, 3 and 4 mM), pH and time of incubation were performed in 50 mM Tris-HCl and 100 mM glycine-NaOH buffers (pH 8,

8.5, 9, 9.5, 10, 10.5, 11, 11.5 and 12). In a typical reaction, the buffer along with enzyme and substrate in a total volume of 3.0 mL were incubated at 37°C for 30 min. However, the reaction mixtures were held at different temperatures (30, 45, 60, 75 and 90°C) and incubation periods (30, 60, 120, 180 and 240 min) whenever required. The reaction was stopped with the addition of 1.0 mL of 1 M-K2HPO4-KOH buffer, pH 10.4. Initial rates of hydrolysis were determined from time-dependent release of p-nitrophenol (€405 = 1.65 × 10

4 M

-1 cm

-1 )

12 . Michaelis-Menten parameters were

determined using Lineweaver-Burk plots. All hydrolytic reactions were performed in triplicate. Bioprecipitation of Cu

2+

Typical reactions containing 50 mM Tris-HCl buffer (pH 11), 15 mM pNPP, 50 ppm or 100 ppm Cu

2+ mono-ion solution (prepared from CuCl2) along

with the enzyme in a volume of 2.0 mL were incubated for 30, 60 or 120 min at 37ºC. After incubation, the reactions were stopped by adding 10 M NaOH (116 µ L) and the reaction mixtures were centrifuged (15000 × g) for 15 min to precipitate metal-phosphate complex. The supernatant was collected and used for analysis by atomic absorption flame spectrophotometer (AAS). Quantification of metal precipitated was calculated as the difference between the initial and final concentration of metal present in the supernatant. Appropriate control reactions without enzyme were also carried out. Percentage of precipitation of metal was calculated by the following formula:

X = (a-b) / a ×100

where X = % of copper precipitation, a = Initial conc. of copper in the aliquot, and b = Final conc. of copper in the aliquot (i.e., in the supernatant) Statistical analysis

The data were statistically analyzed using SPSS 17.0 software (Mean ± SD, n = 3). The values for Cu

2+ precipitation were given as mean of triplicates

with SEM (standard error of the mean). Results and Discussion

Kinetic study of CIAP with pNPP in Tris-HCl buffer (50 mM) and Glycine-NaOH buffer (100 mM)

Effect of pH, substrate concentration and buffer

The activity of ALP like for any other enzyme is known to be pH-dependent. In this investigation, the enzymatic activity as a function of pH and buffer was

INDIAN J. BIOCHEM . BIOPHYS., VOL. 50, FEBRUARY 2013

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investigated. The effects of pH on the kinetics of CIAP-catalyzed pNPP hydrolysis reaction were determined in Tris-HCl and glycine-NaOH buffers. Figures 1a and b showed that CIAP activity was highest at pH 11 in Tris-HCl buffer, whereas in glycine-NaOH buffer the highest activity was recorded at pH 9.5. It is generally presumed that hydrolysis of phosphoryl enzyme is a common rate-determining step of the overall reaction of ALP from E. coli

13 .

Further, Tris buffer itself is reported to be inhibitory to enzyme activity

14, 15 . The rate-determining step at pH

8 was attributed to the non-dissociation of phosphate from the non-covalent enzyme-phosphate complex. On the contrary, it has also been reported that in presence of Tris the rate of hydrolysis of 4-nitrophenyl phosphate is increased at pH 8

16 . There is another

report of increased rate of hydrolysis of ALP with the substrate 4-methylumbelliferyl

17 . However from the

experimental results reported here, it was evident that Tris buffer was not inhibitory even at pH 11. With increasing concentration of substrate, no significant

reduction in the enzyme activity was seen at pH 11; rather the observed activity was much higher than that noticed at other lower pH values i.e., pH 8 and 8.5. At all other pH values i.e., between pH 8.5-11 as well as pH 12, the enzyme consistently showed less activity. Thus, it appeared that the enzyme had two shoulders of functionally stable conformations, one occurring at pH 8-8.5 and the other at pH 11.

The enzyme seemed to be relatively stable catalytically across wide concentration range of the substrate tested i.e., 0.2-4 mM. Such an increase in the pH optima has been reported with ALPs from dog’s intestine, kidney and liver, where higher enzyme activity is recorded at pH 9

18 . The findings in

the present study also showed similarities with the known characteristics of other mammalian ALPs

11,18-20 .

However, the investigations on CIAP in this study revealed extraordinary activity, even at pH 11 with highest Vmax (Fig. 2a). Further, at this alkaline pH of 11, the enzyme also displayed a different temperature optimum at 45ºC, instead of 37ºC. It was also observed that the enzyme activity ceased rapidly beyond 60 min, when the incubation was carried out at 45ºC as opposed to the continuity in the activity seen up to 2 h at 37ºC.

Thus, it appeared that CIAP could undergo subtle conformation changes across the alkaline pH from 8 to 12 in Tris-HCl buffer. The enzyme might be attaining kinetically stable conformations at pH 8.5 and 11, as evident from the experimental observations (Fig. 1a). On the other hand, glycine-NaOH buffer seemed to impart significantly different catalytic pattern to the enzyme with maximum activity at pH 9.5 across all the substrate concentrations tested i.e., 0.2-4 mM. However, it may be still noticed that once again the enzyme exhibited two shoulders of stability one at pH 8-8.5 and the other occurring at pH 9.5 as opposed to at pH 11 observed with Tris-HCl buffer. At all other pH values between pH 8-8.5 and above 9, inhibition of the enzyme activity was noticed.

The effect of substrate concentration on the CIAP activity across a wide range of pH values between 8 and 12 was also observed and shown in Figs 1a and b. Based on the experimental data, 1.5 mM pNPP was found to produce the highest activity at pH 11 in Tris-HCl. However, the enzyme exhibited different catalytic pattern in glycine-NaOH buffer. Although maximum enzyme activity was recorded at pH 9.5, the two shoulders of peak activities corresponded to

Fig. 1—Hydrolysis of pNPP by 1U of CIAP in 3 mL of reaction volume under different concentrations of substrate and varying pH regimes in (a) 50 mM Tris-HCl buffer; and (b) 100 mM glycine- NaOH buffer [The reactions were carried out for 30 min at 37ºC]

CHAUDHURI et al: KINETIC BEHAVIOUR OF CALF INTESTINAL ALP WITH pNPP

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substrate concentrations of 1 mM and 2.5 mM pNPP. The observations in the present study were in sharp contrast to the observations made by Fernley and Walker

11 , who reported inhibition of the enzyme at

these concentrations of the substrate. From the enzyme substrate saturation kinetics shown in Fig. 1a, it was observed that saturation of the enzyme for substrate binding occurred at 1.5 mM, regardless of the

pH 8.5-12 in Tris-HCl buffer. Beyond 1.5 mM pNPP, inhibition in the activity of the enzyme was seen by increasing concentrations of the substrate at all pH values except at pH 11 where steady and stable activity was recorded. Possibly at higher pH of 11, the dissociation of pNP from the pNPP or transfer of the enzyme to another quaternary state might be highly facilitated, although no experimental evidence could be extended. However, such a characteristic feature was not observed with glycine-NaOH buffer.

The relationship between ALP activity and pH varied with the buffer used. The influence of buffer plays a vital role in the enzyme catalytic activity

21 . It

is also reported that buffers having hydroxyl groups participate in the reaction as phosphate acceptors, thereby influencing the kinetic constants of the respective enzyme system

21 . Similarly, the presence

of Tris during the hydrolysis of 4-nitrophenyl phosphate by ALP at pH 8 is reported to increase the rate of 4-nitrophenol liberation with concomitant phosphorylation of Tris

13 . It is also reported that in

alkaline pH, when the rate of hydrolysis is lower, the dephosphorylation of the enzyme takes place at slower pace

13 .

In the present study, the observed increase in the activity at pH 9.5 (glycine-NaOH buffer) and 11 (Tris-HCl buffer) might be due to substrate scavenging of Pi by the hydroxyl groups (-OH) of glycine-NaOH buffer and by the formation of Tris- phosphate respectively. Such an increase in the rate of dephosphorylation and transphosphorylation of the enzyme by Tris-HCl is reported for E. coli ALP with 2, 4-dinitrophenyl phosphate

13 . However, such

assumptions need to be supported by identification of either free phosphate (PO4

¯ ) or Tris-phosphate in

the reaction. Determination of Vmax, Km, Kcat and catalytic efficiency at different pH in 50 mM Tris-HCl and 100 mM glycine-NaOH buffers

Enzyme velocity as a function of substrate concentration often follows the Michaelis-Menten equation:

Velocity (V) = Vmax[S]/[S] + Km

where Km is Michaelis-Menten constant, Vmax is the maximum velocity, [S] is the substrate concentration and V is the initial velocity of the enzyme. Michaelis- Menten equation is applicable for single-substrate enzyme-catalyzed reactions and it involves the formation of a single intermediate complex.

Fig. 2—Effect of pH on (a) Vmax of CIAP, (b) the affinity of CIAP for the substrate, (c) turn over number of CIAP, and (d) catalytic efficiency of CIAP in 50 mM Tris-HCl [Incubation was carried out for 30 min at 37ºC]

INDIAN J. BIOCHEM . BIOPHYS., VOL. 50, FEBRUARY 2013

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For both Tris-HCl and glycine-NaOH buffers over a pH range of 8 to 12, Vmax has been derived from Lineweaver-Burk plot

22 . From the Figs 2 and 3,

it was observed that Vmax and Km varied along with the pH in both the buffers. A low Km value indicates a strong affinity between enzyme and substrate, whereas a high Km value reflects a weak affinity between them

23 . From the experiments, it was found

that the Vmax and Km in Tris-HCl buffer at pH 11 were 3.12 µ moles min

-1 unit

-1 and 7.6 × 10

-4 M,

respectively. It indicated that although the rate of hydrolysis was maximum at pH 11, the affinity of the enzyme for the substrate was at its least, a reflection of possible subtle conformational changes of the enzyme. Further, there could be reorganization of binding sites at this alkaline pH.

In the case of glycine-NaOH, it was found that at pH 9.5 the Vmax and Km were 1.6 µ moles min

-1 unit

-1

and 4 × 10 -4

M, respectively. From Fig. 2 (a-c) and 3 (a-c), it can be deciphered that the enzyme was at its best with respect to pH optima in Tris-HCl at 11 and glycine-NaOH at 9.5. However, significant differences were noticed in its affinity to the substrate i.e., 7.6 × 10

-4 M in Tris-HCl buffer, compared to

4 × 10 -4

M in glycine-NaOH buffer. CIAP is reported to have Km value of 9.6 × 10

-4 M for phenyl

phosphate 18

while it is found to be 3 × 10 -2

M for β-glycerophosphate for rat intestinal-mucosal ALP24. In contrast, in the current study, the Km values observed were several orders of magnitude lesser than what is reported in earlier studies and the higher affinity reported here could be partly due to the purified enzyme used in this investigation

16,24 .

The highest turnover number with respect to Tris-HCl and glycine-NaOH was found be 82.98 and 42.55 s

-1 respectively, which was also reflected in

the highest specific activity of the enzyme as seen in Figs 2c and 3c. The highest catalytic efficiency of 18187.1 M

-1 s

-1 was obtained at pH 8 for

Tris-HCl, while it was 106375 M -1

s -1

for glycine- NaOH buffer at pH 9.5 (Figs 2d and 3d). Interestingly, the catalytic efficiency in Tris-HCl was quite comparable at pH regimes 8, 9, 9.5, 10 and 11 (Fig. 2d), while a significant peak was noticed at pH 9.5 in glycine-NaOH buffer (Fig. 3d).

Effect of temperature on CIAP activity in Tris-HCl and glycine-NaOH buffers

From the experiment (Figs 4a and b), it was observed that catalytic hydrolysis of pNPP reaction varied with respect to different temperatures in both

Fig. 3—Effect of pH on (a) Vmax of CIAP , (b) the affinity of CIAP for the substrate, (c) turn over number of CIAP and (d) catalytic efficiency of CIAP in 100 mM glycine-NaOH [Incubation was carried out for 30 min at 37ºC]

CHAUDHURI et al: KINETIC BEHAVIOUR OF CALF INTESTINAL ALP WITH pNPP

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Tris-HCl and glycine-NaOH buffer. Earlier, it is shown that the range of temperature requirement falls between 20 to 40ºC for 4-methylumbelliferyl phosphate for CIAP

11 . In rabbit liver ALP,

the optimum temperature registered is 45ºC 25

. Experiments on free and immobilized CIAP in phosphate buffer at pH 7 have shown reduction in the enzyme activity above 40ºC

26 . In the present

study, the maximum activity of the enzyme was found at 45ºC in both Tris-HCl and glycine-NaOH buffers. In Tris-HCl buffer, between 30ºC and 45ºC the activity was found to be twice of the initial value. Further, the enzyme registered at least 5-10 folds more activity in Tris-HCl buffer compared to glycine-NaOH buffer, when examined across the temperature range of 30 to 90ºC.

Conforming to the established notion that the velocity of an enzyme reaction increases with an increase in temperature up to a maximum and then declines, the enzyme CIAP employed in the current investigation displayed a peak activity at 45ºC, regardless of the buffer used. In addition, the enzyme displayed a two-fold increase in the activity when the temperature was raised from 37ºC to

45ºC. The calf intestine being in a ruminant contains many bacteria which are involved in fermentation process and hence there could be periodical bursts in intestinal temperature beyond 37ºC

27 . The inherent

temperature optimum of 45ºC recorded in this study indicated evolutionary significance for the enzyme to operate under such perturbances in the temperature of intestine. Effect of incubation period on CIAP activity at 37°C and 45ºC in Tris-HCl and Glycine-NaOH buffers

It was found that with increasing period of incubation, an increase in the activity of the enzyme was seen in Tris-HCl buffer under standard conditions of reactions. The activity reached a maximum at 120 min, followed by a steady decline thereafter (Fig. 5a). On the contrary, in glycine-NaOH buffer, the enzyme displayed decline in the activity during the incubation time beyond 30 min (Fig. 6a). Further, the activity was several folds lesser than that noticed under conditions of Tris-buffer. Since maximum activity of the enzyme was observed at 45ºC in both Tris and Glycine buffers, activity of the enzyme was checked at different time intervals at

Fig. 4—Effect of temparature on CIAP activity in (a) 50 mM Tris-HCl buffer at pH 11 and (b) 100 mM glycine-NaOH buffer at pH 9.5 [The reactions were carried out with 1.5 mM pNPP for 30 min]

Fig. 5—Effect of incubation period on CIAP activity (a) at 37ºC and (b) at 45ºC in 50 mM Tris-HCl at pH 11 [The reactions were carried out with 1.5 mM pNPP]

INDIAN J. BIOCHEM . BIOPHYS., VOL. 50, FEBRUARY 2013

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45ºC in both buffers. In Tris-buffer, the maximum activity of CIAP was observed at 60 min (Fig. 5b) and in Glycine buffer, it was obtained at 30 min (Fig. 6b). Such an extended activity both at 37ºC and 45ºC makes the enzyme suitable for industrial applications such as bioremediation of industrial effluents etc

28,29 .

Application of CIAP and pNPP for precipitation of heavy metal copper (Cu

2+ ) from CuCl2 solution

It was observed that CIAP was capable of precipitating Cu

2+ from both the concentrations of

Cu 2+

mono-ion solution employed i.e., 50 ppm and 100 ppm at pH 11. The highest precipitation obtained for 50 ppm initial concentration was 64.3% (after 120 min incubation period) (Fig. 7). On the other hand, for an initial concentration of 100 ppm, the precipitation was only 47.4% (Fig. 7) during the same incubation period. Thus, the observations on the precipitation of copper demonstrated the ability of inorganic phosphate (Pi) driven bioprecipitation catalyzed by CIAP.

Conclusion

The kinetic behaviour of the enzyme under different substrate concentrations, pH conditions, temperature and period of incubation time indicated that the enzyme might undergo subtle conformational changes, leading to significant changes in the catalytic activity, which perhaps cannot be accounted for by pure random collisions and other physical and chemical phenomenon. Such changes probably can be interpreted by in silico analysis. Since CIAP is capable of precipitating heavy metals via Pi generation from a variety of substrates as demonstrated in this study for precipitating Cu, it may find use for the treatment of industrial effluents. However, further studies are required for exploring the utility of the enzyme in immobilized state on polymeric fibers or membranes for repeated use during effluent treatment in the waste management plants. Acknowledgement

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Fig. 6—Effect of incubation period on CIAP activity (a) at 37ºC and (b) at 45ºC in 100 mM glycine-NaOH at pH 9.5 [The reactions were carried out with 1.5 mM pNPP]

Fig. 7—Percentage of precipitation of Cu

2+ for two different

concentrations of single ion solution [Mean ± SEM; n = 3].

CHAUDHURI et al: KINETIC BEHAVIOUR OF CALF INTESTINAL ALP WITH pNPP

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