Biokemistri Nigerian Society for Experimental Biology ISSN: 0795-8080 Vol. 19, Num. 2, 2007, pp. 43-48

Biokemistri, Vol. 19, No. 2, Dec, 2007, pp. 43-48

Cofactor interactions in the activation of tissue non-specific alkaline phosphatase: Synergistic effects of Zn²⁺ and Mg²⁺ ions

Femi J. Olorunniji*, Adedoyin Igunnu, Joseph O. Adebayo, Rotimi O. Arise and Sylvia O. Malomo

Department of Biochemistry, University of Ilorin, P.M.B. 1515 Ilorin, Nigeria
* corresponding author. Email: femijohn@gmail.com

Received 19 March 2007

Code Number: bk07007

Abstract

The interactions of Mg²⁺ and Zn²⁺ ions in the activation of non-specific tissue alkaline phosphatase were investigated using crude extracts of rat kidney. Activation of alkaline phosphatase by the metal ions was accompanied by changes in the kinetic parameters of p-nitrophenylphosphate hydrolysis. The results suggest some synergistic interactions between Mg²⁺ and Zn²⁺ ionsin promoting the hydrolysis of p-nitrophenylphosphate by alkaline phosphatase. The results show that assays of alkaline phosphatase activity in homogenised tissue samples will give better responses if both Mg²⁺ and Zn²⁺ ions are included in the reactions 

Keywords:Alkaline phosphatase; kinetics; Enzyme-cofactor interaction; synergism

INTRODUCTION

The roles of metal ions in metalloenzymes include direct participation in catalysis, stabilization of protein structure and regulation of enzymatic activity. Membrane alkaline phosphatase (ALP) is a metal-containing enzyme that serves as a good model for the study of metal ion interactions in enzyme catalysis. Native E. coli ALP contains three metal ion binding sites (two Zn²⁺ sites and one Mg²⁺ site), and studies on their roles and interrelationships have provided some insights into the mechanism of the enzyme¹. E. coli ALP is a zinc metalloenzyme, which can be activated by magnesium ion². Removal of the Zn²⁺ leads to loss of catalytic activity while its replacement by other divalent cations (Mn²⁺, Co³⁺, Cd²⁺, and Cu²⁺) resulted in lower maximal activity². It has been shown that while low concentrations of Mg²⁺ stimulated the refolding of E. coli ALP, high concentration actually inhibited its reconstitution into the active form³. This suggests that Mg²⁺ mediates stabilization and destabilization of the catalytically active structure of ALP at low and high concentrations respectively^4,5. In E. coli ALP, Mg²⁺ is thought to have a regulatory effect on the expression of catalytic activity and maintenance of structural integrity of the enzyme¹. The specific binding of Mg²⁺ to apo-ALP depends on both pH and the cooperative effects of Zn²⁺ binding. Mg²⁺ alone does not confer catalytic activity on ALP, but it does regulate the Zn²⁺-induced restoration of activity and perhaps, structural integrity of the metal-binding loci^1,6.

Mammalian ALPs are glycoproteins that are present as different isoenzymes in several tissues including bone, intestine, kidney, and placenta. Like their E. coli counterpart, they are zinc-metalloenzymes that can be activated by Mg²⁺ ions; both ions being essential for catalysis and structural stability. The human genome, like other mammalian systems contains four ALP loci, one coding for the tissue nonspecific ALP (TNAP) expressed in a variety of organs such as liver, bone, kidney, etc. and three tissue-specific ALP (TSAP) genes coding for the intestinal AP (IAP), placental ALP (PLAP), and germ cell ALP (GCAP). TNAP shows approximately 50% sequence similarity with the TSAP isozymes. Mammalian ALPs show 25-35% sequence identity with the E. coli enzyme in those regions of the protein assuming a-helix and b-strand secondary structures, and critical for catalysis. The catalytic residues, i.e. Asp91, Ser92, Arg166, and ligands coordinating the divalent metal ions (Zn²⁺ and Mg²⁺) are all conserved². These structural similarities suggest that mammalian ALPs may catalyse hydrolysis of phosphate monoesters via a similar mechanism as the E. coli enzyme. 

Structural and functional aspects of the role of divalent cations have been extensively studied in E. coli ALP. Such studies have been facilitated by the availability of several X-ray structures of E. coli ALP. In contrast, less direct information is available on the structure and mechanism of mammalian ALPs. Owing to the practical importance of assaying tissue ALP activities in applied biochemical studies involving use of animals⁷ and the mechanistic interest of the subject, we have been studying the effect of cofactors and inhibitors on the catalytic properties of mammalian ALPs^5,8. Kinetic analysis shows that high concentrations of Mg²⁺ inhibit tissue non-specific ALP in crude extracts by decreasing V_max of pNPP hydrolysis⁵. Here we report a synergistic interaction of Mg²⁺ and Zn²⁺ ions in the activation of tissue non-specific ALP. The implications for the determination of alkaline phosphatase in crude tissue extracts are discussed. 

MATERIALS AND METHODS

Reagents, chemicals, and enzyme source

ALP substrate, the sodium salt of p-nitrophenylphosphate (pNPP) was obtained from Sigma Chemical Company, St. Louis, US. MgCl₂ and ZnCl₂ were products of British Drug House, UK. All other chemicals used in this study were of high quality research grade. The tissue non-specific ALP used here was obtained from rat kidney. Fresh kidney tissue from Rattus Norvegicus was homogenized in 0.25 M sucrose solution at 4 °C and the crude homogenate was centrifuged at 4000 rpm for 20 minutes at the same temperature. The supernatant was collected and dispensed in Eppendorf tubes and stored frozen. Fresh aliquots were used each day for the experiments described in this report. It is generally believed that to perform a large number of kinetic measurements under constant conditions it is more important to have large stabilized enzyme preparations than small preparations with a higher activity⁹. Thus, we did not attempt further purification since the enzyme is unusually stable even in impure crude preparations¹⁰. The key experimental results obtained with the crude ALP extract were validated with homogenous calf intestinal ALP obtained from New England Biolabs. 

Determination of alkaline phosphatase-catalysed hydrolysis of pNPP

Alkaline phosphatase activity was measured by the rate of hydrolysis of appropriate concentrations of p-nitrophenylphosphate (pNPP) at 25 °C in 0.1 M Na₂CO₃/NaHCO₃ buffer, pH 10.1 as previously described⁹. Enzyme activity is expressed as the mmol of p-nitrophenol released per minute. Reaction mixtures containing the enzyme source and buffers in the presence of the appropriate metal ions were kept at 25 °C for 10 minutes. In all assays, 100 ml of the crude enzyme preparation equivalent to 0.5 mg total protein were added to catalyse the hydrolysis of pNPP. Reactions were initiated by the addition of the appropriate concentration of the substrate, pNPP. Incubation was allowed for 10 minutes before stopping the reaction by the addition of 0.1 M KOH. The absorbance was read at 400 nm against a blank of the buffered substrate on a Specronic-21 UV-Vis spectrophotometer and the corresponding activities recorded. All measurements of reaction rate were performed in triplicate. Protein concentration was determined using Biuret method¹¹.

RESULTS

Activation of alkaline phosphatase by Mg²⁺ and Zn²⁺    

An analysis of the roles of Zn²⁺ and Mg²⁺ ions in the activation of ALP was carried out by investigating the kinetics of pNPP hydrolysis in the presence of 0, 2, 4 and 8 mM of the two metal ions separately (Figure 1). In experiments to determine kinetic constants, activity was monitored by measuring the absorbance change at varying substrate concentrations (0.2–2 mM pNPP) in the presence of varying metal ion concentrations. The effects of Mg²⁺ and Zn²⁺ on the K_m for pNPP and V_max values were obtained from Lineweaver-Burk plots (Table 1).Within the range of substrate concentration examined, the hydrolysis of pNPP by ALP followed saturation kinetics (Figure 1A).

Kinetic analysis showed that a progressive decline in activation of ALP occurred as Mg²⁺ concentration was raised from 2 mM to 8 mM as observed by the progressive decrease in the maximum reaction rate V_max. The activation of alkaline phosphatase by Mg²⁺ was also accompanied by a biphasic change in the Michaelis constant (K_m). Increasing Mg²⁺ concentration from 2 mM to 4 mM resultedin a decrease in the K_m for pNPP. However, the K_m was higher at 8 mM Mg²⁺ concentration.

The activation of ALP by Zn²⁺ followed a pattern similar to that of Mg²⁺ in affecting both the V_max and the K_m (Figure 1B). However, the effect of Zn²⁺ on the K_m for pNPP did not show the biphasic pattern seen with Mg²⁺. There was a consistent decrease in the K_m value as the Zn²⁺ concentration was increased from 0 to 8 mM (Table 1).

Pre-incubation of alkaline phosphatase with Mg²⁺ and Zn²⁺

The results shown in Figure 1 and Table 1 indicate that Mg²⁺ ion at higher concentrations inhibit pNPP hydrolysis. This could be due to occupancy of the Zn site that is essential for catalysis by the higher concentration of Mg²⁺. Hence, we sought to investigate this possibility by determining how pre-incubation of the enzyme with Zn²⁺ and Mg²⁺ ions prior to the addition of the substrate affects catalytic activity. Each metal ion (4 mM) was added to the appropriate amount of the enzyme for different incubation periods (0, 5, 10, 15, 30 and 60 minutes) before adding 1.0 mM pNPP for 10 minutes. Figure 2 shows the effect of separate pre-incubation of ALP with 4 mM Zn²⁺ and 4 mM Mg²⁺ on phosphatase activity. The results show that the effects of Zn²⁺ and Mg²⁺ in activating ALP were time-dependent. The activity of ALP increased progressively when pre-incubated with Zn²⁺ from 0 to 60 minutes. For Mg²⁺ however, the activity of ALP increased progressively with time of pre-incubation up to 10 minutes after which a progressive decrease in activity was observed.

Synergistic interaction of Mg²⁺ and Zn²⁺ on alkaline phosphatase activity

Results presented in Figures 1 and 2 show that both Mg²⁺ and Zn²⁺ ions are required for ALP activation. Hence, we sought to determine if any synergistic interactions exist between both cofactors in the activation of ALP, and to determine the optimal concentrations of both ions for full activity. The effects of Mg²⁺ and Zn²⁺ ions on ALP activity were investigated by determining the rate of hydrolysis of 1.0 mM pNPP under different conditions. The conditions are: Control (0 mM Mg²⁺, 0 mM Zn²⁺), 2 mM Mg²⁺, 4 mM Mg²⁺, 2 mM Zn²⁺, 4 mM Zn²⁺,2 mM Mg²⁺ & 2 mM Zn²⁺, 2 mM Mg²⁺ & 4 mM Zn²⁺, 4 mM Mg²⁺  & 2 mM Zn²⁺, and 4 mM Mg²⁺ & 4 mM Zn²⁺. Significant differences between the rates of pNPP hydrolysis obtained under the different conditions were determined at 5% confidence level using the Duncan’s Multiple Range Test¹². The results of this experiment are shown in Figure 3 and the pattern indicates a synergistic interaction between the two metal ion cofactors.

DISCUSSION

It has been established that E. coli alkaline phosphatase contains three classes of metal binding sites in which Zn²⁺ occupies the catalytic and structural sites, while Mg²⁺ ions are bound at the regulatory site^1,13. The activation of ALP-catalysed hydrolysis of pNPP observed in this study by the two metal ions is exerted via both V_max and K_m effects. The decline in activation at 8 mM Mg²⁺ observed here is consistent with the observation in our earlier study that high concentration of Mg²⁺ inhibits the non-specific ALP from rat liver⁵. It was shown that Mg²⁺ acts as an activator within optimal concentrations but became inhibitory at higher concentrations. One possible explanation for this set of results is that excess Mg²⁺ ions displaced Zn²⁺ from the catalytic site since both metal ions can bind to the same site. The effect of Mg²⁺ concentrations on the Michaelis constant (K_m) suggests that Mg²⁺ activates kidney ALP by increasing the affinity of the enzyme for pNPP. This agrees with the report that the binding of Mg²⁺ to kidney ALP promotes a conformational change⁹, which causes the displacement of the substrate into the vicinity of zinc-coordinated water molecule, thus leading to increased affinity of the enzyme for its substrate. The concentration-dependent activation of kidney ALP by Zn²⁺ agrees with the findings of Bosron et al. (1977)¹. The observation that Zn²⁺ affects K_m may suggest that Zn²⁺ induces its activation effect on ALP through its interaction with the free enzyme. The observed increase in V_max due to increasing Zn²⁺ concentration most likely reflects the catalytic requirement for the metal ion in phosphate ester hydrolysis¹⁴.

Pre-incubation of ALP activity with the metal ion cofactors led to increase in the hydrolysis of ALP.  Hence, the metal ions probably mediate certain time-dependent effects that result in stimulation of activity. Such progressive activation is characteristic of conformational changes at or around the active site and may involve optimizing substrate binding or the actual steps of catalysis. A structural role for Mg²⁺ has been proposed in earlier studies^5,13. The inhibition that results after prolonged incubation of ALP with Mg²⁺ may be due to displacement of Zn²⁺ from its binding site on the enzyme¹⁵.

Earlier works showed that ALP contains two Mg²⁺ binding sites and four Zn²⁺ binding sites in its active site^14,16. Thus, the synergistic interaction observed between Mg²⁺ and Zn²⁺ in activating ALP (Figure 3) suggest that both metal ions are required in a defined optimal ratio. The scope of this study is not sufficient to ascertain the exact ratio of the two metal ions required for optimal hydrolase activity. Mg²⁺ is usually employed as the only cofactor in routine ALP assays in clinical chemistry as well as in toxicological investigations^7,8,14. However, the results obtained in this study suggests that better responses are likely to be obtained in such assays if carried out in the presence of both Mg²⁺ and Zn²⁺ at appropriate concentrations.

REFERENCES

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13. Anderson, R.A., Bosron,W.F., Kennedy, F.S. and Vallee, B.L. (1975) Role of Magnesium in Escherichia coli alkaline phosphatase. Proc. Natl. Acad. Sci. U.S.A.  72: 2989-2993.
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