Hydrolysis of glucose-6-phosphate in aged, acid-forced hydrolysed nanomolarinorganic iron solutions—an inorganic biocatalyst?{

Xiao-Lan Huang{§*a and Jia-Zhong Zhang§b

Received 24th June 2011, Accepted 22nd August 2011

DOI: 10.1039/c1ra00353d

Phosphate ester hydrolysis is one of the most important chemical processes in biological systems.

Although catalysis by the natural phosphoesterases, e.g., purple acid phosphatase (PAP) and its

biomimetics, are well known in biochemistry, it has been reported that some metals and mineral

phases can significantly facilitate the hydrolysis of phosphate ester. Here we report for the first time

that aged, acid-forced hydrolysed nanomolar inorganic iron solutions significantly promoted the

hydrolysis of glucose-6-phosphate (G6P), and that the reaction kinetics followed the Michaelis–

Menten equation. The catalysis was inhibited by tetrahedral oxyanions in an order of WO4 . MoO4

. PO4. The newly formed oxo-bridge or hydroxo-bridge during the iron-aging process might

contribute to this biocatalytic effect, though the detailed mechanism is still unclear. Further studies

are needed in order to understand the (hydr)oxo-bridged Fe–Fe structure in water and its role in

organic phosphorus transformation. This catalyst might be one of many ubiquitous sets of inorganic

enzymes yet to be discovered in nature that act as a bridge between the inorganic and organic worlds,

and would have played a critical role in the origin of life.

1. Introduction

Glucose-6-phosphate (G6P), an example of a phosphate ester, is

glucose sugar phosphorylated on carbon 6, and widely dis-

tributed in nature, including water and soil.1–3 As an essential

process of life,4–8 G6P hydrolysis is catalyzed by various

enzymes, including phosphoesterases9–15 such as purple acid

phosphatase (PAP). It is also known that their biomimetics, i.e.,

metals, especially iron, chelated to special organic ligands, can

significantly promote the hydrolysis of phosphate ester.16–28

Moreover, it has been reported that some metals29–32 and

mineral phases33–37 can significantly facilitate the hydrolysis of

ester phosphate, including nucleoside phosphates. Therefore,

understanding the behavior of phosphate ester hydrolysis is

important for biology and biogeochemistry as it is relevant to

phosphate availability in the environment.2,38–43

Recently, we discovered that hydrolysis of G6P is significantly

promoted by aged, acid-forced hydrolysed nanomolar iron

solutions, and the presence of some tetrahedral oxyanions in

solution act as inhibitors in an order of WO4 . MoO4 . PO4.

The kinetics of G6P hydrolysis in these nanomolar inorganic

iron solutions can be described by the Michaelis–Menten

equation. Here, we report these interesting phenomena and

hypothesize that the (hydr)oxo-bridged Fe–Fe structure in the

aged iron solution contributes to the observed catalytic effect.

2. Experimental

Deionized water (DIW), used for preparing standards, reagents,

and aged iron solutions, was purified first with a distilling unit

and then by a Millipore Super-Q Plus water system that

produced water with a resistivity of 18 MV cm. All reagents

and aged iron solutions were stored in polypropylene bottles (or

containers) that were immersed in a 10% HCl solution overnight,

followed by rinsing three times with DIW and then drying at

60 uC in an oven for 5–10 h prior to their use.

All chemicals used were of analytical grade (AR or GR), and

used as received. Glucose-6-phosphate (D(+)-glucopyranose

6-phosphate sodium salt, G-6-P Na, C6H12NaO9P, F. W.

282.12), glycerol 2-phosphate (b-glycerophosphate disodium salt

hydrate, G2P, C3H7Na2O6P, F. W. 216.04), ribose-5-phosphate

(D-ribofuranose 5-phosphate disodium salt, R5P, C5H9Na2O8P?

xH2O, F. W. 274.07), and fructose 1-phosphate (D-fructose

1-phosphate sodium salt, F1P, C6H11O9PNa2, F. W. 304.10),

aCIMAS, Rosenstiel School of Marine and Atmospheric Science,University of Miami, Miami, Florida, 33149, USA.E-mail: xiaolan.huang@ymail.com.; Fax: 1-513-252-2332;Tel: 1-513-569-7409bOcean Chemistry Division, Atlantic Oceanographic and MeteorologicalLaboratory, National Oceanic and Atmospheric Administration (NOAA),Miami, Florida, 33149, USA{ Electronic supplementary information (ESI) available: Fig. 1: timecourse of formation of phosphoantimonylmolybdenum blue complexfrom phosphate released during hydrolysis of 20 mM G6P in an aged-4month, 1 mM iron solution at room temperature (22 ¡ 2 uC) at differenthydrolysis times. Table 1: effect of tris-buffer solution on the G6Phydrolysis in an aged-14 month, 1 mM iron solution. See DOI: 10.1039/c1ra00353d{ Present address: Pegasus Technical Services Inc., 46 E Hollister St.,Cincinnati, OH 45219, USA§ X.-L. H. instigated this study and performed the experiments, andwrote the draft of the manuscript. X.-L. H. and J.-Z. Z. designed theexperiments, and prepared the final manuscript.

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were purchased from Sigma and stored in a freezer (220 uC). A

50 mM stock solution for each organic phosphorus compound was

made individually, stored in a refrigerator at 4 uC, and diluted to

suitable concentrations for daily hydrolysis experiments.

Inorganic phosphate (IP) derived from the hydrolysis of G6P

was determined by a modified method of Murphy and Riley in

which the final total pHT of the test solution (sample mixed with

reagents) was 1.0 (H/Mo = 70).44 An ammonium molybdate

reagent was prepared by mixing 2.4 g of ammonium molybdate

((NH4)6MO7O24?4H2O, Merck, GR), 25 mL of concentrated

sulfuric acid (H2SO4, 96–98%, J.T. Baker), and 50 mL of 0.3%

antimony potassium tartrate (K(SbO)C4H4O6)2?H2O, Fisher)

solution and diluting the mixture to 1 L with DIW. An ascorbic

acid solution was prepared daily by dissolving 1 g of ascorbic

acid (C6H8O6, Aldrich, AR) in 100 mL of DIW. Prior to sample

analysis, a colour reagent was prepared by mixing equal volumes

of the molybdate reagent with the ascorbic acid solution. One ml

of the colour reagent was then added to 4 ml of the sample and

mixed. After 10 min, absorbance of phosphoantimonyl-molyb-

denum blue was measured at 890 nm with background

corrections at 780 to 1020 nm by a Hewlett-Packard 8453 UV-

visible spectrophotometer.

2.1. Preparation of aged iron salt solutions

Since acidic environments significantly reduce the rate of

iron(III) hydrolysis,45–48 six series of inorganic iron(III) salt

solutions were made by rapid dilution under acidic conditions to

nanomolar concentrations. Five different iron salts and a

commercial iron standard solution for atomic absorption

spectrophotometry (J.T.Baker, prepared by dissolving metal Fe

in 0.3 M HNO3 to yield a concentration of 1000 mg ml21) were

used. The total iron concentration in the solutions used for G6P

hydrolysis experiments ranged from 1 to 10 000 nM, and the

maximum aging time was 16 months.

2.1.1. Aged 16.5 nM Fe solutions. A 10 mM Fe solution was

prepared by dissolving 2.02 g of Fe(NO3)3?9H2O (J.T.Baker, AR,

F. W. 404.00) or 1.96 g of Fe(SO4)2(NH4)2?6H2O (EM Science,

GR, F. W. 392.14) in 500 ml of dilute HNO3 solution (pH 3.0).

This solution was immediately diluted 10 times with DIW. Such

dilution (a 10 to 1 ratio) by DIW was repeated with newly

prepared, an order of magnitude lower concentration of Fe

solutions until the Fe concentration reached nM levels. All

sequential dilutions were processed within 10 min to minimize Fe

hydrolysis at higher concentrations. A 500 nM Fe-EDTA solution

was also prepared by first diluting the above mentioned 10 mM

Fe(NO3)3 10 times with DIW and adding EDTA at a molar ratio

of 1 : 4 (Fe : EDTA) to make a 1 mM Fe-EDTA solution and

subsequent step-wise dilutions (10 times dilution at each step). After

3 months, a 50 nM Fe solution was made from the aged 500 nM Fe

solution by dilution with DIW. After another 2 months of aging,

2 mM of NaHCO3 was added to the 50 nM aged iron solution, to

yield a final concentration of 16.5 nM iron in 0.67 mM NaHCO3.

The pH of the aged iron solution was adjusted to 6.3 ¡ 0.1 by

adding either 0.1 M HCl or 0.1 M NaOH or 20 mM NaHCO3 five

times during the aging process. A noticeable catalytic reactivity

was detected after 6 months of aging. The final measurement was

made at either 14 or 16 months.

2.1.2. Aged 1000 and 10 000 nM Fe(NO3)3 solutions. A

100 mM Fe solution was prepared by dissolving 2.02 g of

Fe(NO3)3?9H2O (Riedel-de Haen, AR, F. W. 404.00) in a dilute

HNO3 solution (pH 3.0). This solution was then diluted with

DIW (pH 6.2) step-by-step (10 times dilution at each step) to

produce 1000 and 10 000 nM Fe solutions. The dilution process

was completed within 10 min. The pH of these iron solutions was

adjusted to 6.3 ¡ 0.1 after aging for 140 to 300 days. The

hydrolysis rate of G6P was measured at 6 and 10 months. All

inhibition experiments and buffer effects were made with this

aged 1000 nM Fe solution.

2.1.3. Aged 1 to 5000 nM ferric solutions. This series of ferric

solutions was prepared from J.T.Baker’s iron standard solution

(1000 mg ml21 Fe(NO3)3). A 1 mM Fe(III) solution was made by

diluting the stock Fe solution with diluted HNO3 (pH 3.0). A

5 mM solution was made by step-wise dilution (2–10 6 dilution

at each step by DIW) from the 1 mM Fe solution. A series of 50–

5000 nM Fe solutions were prepared by diluting the 5 mM Fe

solution. A series of 1–10 nM Fe solutions were prepared by

diluting the 50 nM Fe solution. These dilutions were completed

within 10 min. The pH of these Fe solutions was maintained at

6.3 ¡ 0.1 by adding dilute HCl or NaOH.

2.1.4. Aged 2 and 10 nM FeCl3 solutions. A 10 mM FeCl3solution was prepared by dissolving 0.811 g of FeCl3 (J.T.Baker,

AR, F. W. 162.20) in 500 ml dilute HCl (pH 3.0), and then

diluting it with DIW (pH 6.20) step-by-step (10 6 dilution at

each step) to a 500 nM Fe solution (within 3 min). After aging

for 6 months, 2 and 10 nM Fe solutions were made from the

aged 500 nM Fe solution, and the pH of the aged iron solutions

was adjusted to 6.3 ¡ 0.1.

2.1.5. Aged 10 and 100 nM FeCl3 solutions. A 10 mM FeCl3solution was prepared by dissolving 0.811 g FeCl3 (Riedel-de

Haen, AR, F. W. 162.20) in 500 ml dilute HCl (pH 5.0), which

was then diluted step-by-step by DIW (pH 6.20) to make a final

concentration of 10 and 100 nM (procedure described in Section

2.1.2). The pH of these nM iron solutions was adjusted to 6.3 ¡

0.1 after 100 days.

2.1.6. Aged 10 and 100 nM Fe(ClO4)3 solutions. A 10 mM

Fe(ClO4)3 solution was prepared by dissolving 1.77 g Fe(ClO4)3?

xH2O (Aldrich, GR, low chloride, Cl , 0.005%, F. W. 354.2) in

500 ml dilute HCl (pH 5.0), and then diluting it step-by-step

(10 6 dilution at each step) by DIW (pH 6.2) to make a final

concentration of 10 and 100 nM (procedure described in Section

2.1.2). The pH of these iron solutions was adjusted to 6.3 ¡ 0.1

after 100 days.

2.2. Measurement of hydrolysis rate of G6P

The increase in concentration of IP due to hydrolysis of G6P was

measured at suitable time intervals with a modified colorimetric

method after G6P was added to the aged iron solution. The

concentration of G6P at a given time, t, was calculated using

[G6P]t = [G6P]0 2 [IP]t, where [G6P]0 is an initial concentration

of G6P and [G6P]t and [IP]t are the concentrations of G6P and

IP at time t, respectively. A small amount of IP was initially

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present in the G6P as an impurity, and was subtracted as a

blank. The first-order hydrolysis rate constant, k (s21), was

determined from the slope of the linear portion of a log[G6P]t vs.

time (s) plot. The typical correlation coefficient of the linear

fitting was 0.99*; the duration of the hydrolysis experiments

ranged from 24 to 48 h (with the exception of the control). The

time courses of formation of phosphorantimonylmolybdenum

blue complex from phosphate released from hydrolysis of 20 mM

G6P in an aged (4 months) 1000 nM iron solution at room

temperature (22 ¡ 2 uC) at different hydrolysis time (0, 1, 3, and

6 h) is presented in the ESI Fig. 1.{In the early stages of these experiments, either azide or CHCl3

was added to the aged iron solution to test for potential

microbial contamination. No significant difference was observed

between inhibitor-added and no-inhibitor-added experiments. In

the subsequent experiments reported here, none of the microbial

inhibitors were used.

2.3. Inhibition experiments with different tetrahedral oxyanions

All inhibition experiments were conducted with the 10 month

aged 1000 nM Fe(NO3)3 solution. The tetrahedral oxyanions

included orthophosphate (PO4), molybdate (MoO4) and tung-

state (WO4), and were added as a stock solution into the aged

iron solution before the G6P solutions were introduced. IP

concentration was measured at different times and the hydrolysis

rate constant was calculated as described in the previous section.

3. Results and discussion

3.1. Promotion effect of sugar phosphate hydrolysis in aged iron

solutions

G6P hydrolysis without enzymes is a slow process in DIW and

fresh nanomolar inorganic iron solutions. The concentration of

IP in the solution containing 20 mM G6P in pure water (DIW,

pH 6.2) was 0.42 ¡ 0.02 mM initially (t = 0), and increased

slowly with time. The IP concentration was 0.59 ¡ 0.08 mM at

24 h, and reached up to 2.4 mM after 9 days at room temperature

(22 ¡ 2 uC). The IP concentrations in the fresh nanomole

inorganic iron solutions (0.5 to 50 nM Fe(NO3)3) was 0.82 ¡

0.11 mM after 24 h. However, after G6P was added into the aged

nanomolar inorganic iron salt solutions, made by acid-forced

hydrolysis, the IP was rapidly released. For example, after an

initial 20 mM of G6P was added into a 14 month aged 16.5 nM

Fe(NO3)3 solution (pH=6.3) at room temperature, IP was

rapidly increased due to the hydrolysis of G6P. The concentra-

tion of IP at 1, 3 and 6.7 h was 2.8, 6.4 and 10.6 mM, respectively.

Therefore, the hydrolysis of G6P was significantly promoted in

the aged inorganic iron solution (Fig. 1a). Like metal ions as well

as natural and biomimetic enzymes, the kinetics of G6P

hydrolysis in the aged iron solution can be described as the

pseudo-first-order reaction (Fig. 1b).22,23,29,30,32,49–51 In this

experiment, the decrease in G6P concentration, [G6P]t, due to

its hydrolysis can be expressed as a function of hydrolysis

time, t, as

log[G6P]t = 20.0000131t 2 4.718 (r2 = 0.999) (1)

where [G6P]t is in M and t is in seconds.

Fig. 1 Hydrolysis of G6P in a 16.5 nM Fe(NO3)3 solution aged for 14

months at room temperature (22 ¡ 2 uC). (a) Concentration of IP and

G6P during 20 mM G6P hydrolysis. (b) Pseudo first-order reaction

kinetics of G6P. (c) Double reciprocal (initial velocity and initial

concentration of G6P) plot (Lineweaver–Burk plot) of G6P from 5 to

250 mM. (d) Initial velocity of G6P hydrolysis (v) as a function of the

initial concentration of G6P.

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The corresponding reaction rate constant (k) was 3.02 61025 s21, and the half-life (t1/2) was 6.38 h. In contrast, the

average reaction rate constant in DIW and these fresh unaged

nanomolar inorganic iron solutions was 1.53 6 1027 and 1.98 61027 s21, respectively, and the half-life was 1255 and 1100 h,

respectively. It is noted that the hydrolysis rate constant in the

aged iron solution was much higher than previously reported

rates in the presence of the millimole metals.29–32

The first-order reaction kinetics predicts a constant half-life of

reaction regardless of the initial reactant concentrations.

Consequently, the initial hydrolysis velocity of G6P (vo) is the

product of the hydrolysis rate constant (k) multiplying the initial

concentration of G6P ([G6P]0). However, the half-lifes and the

hydrolysis rate constants at different initial G6P concentrations

were not constant. For G6P hydrolysis at an initial concentra-

tion of 100 mM, the reaction rate constant was 8.83 6 1026 s21,

and the half-life was 21.8 h. This suggests that there was some

intermediate product formation related to the initial concentra-

tion of G6P and a constant equilibrium of intermediate products

might be established during the hydrolysis process, after the

initial G6P was introduced into the aged iron solution. Similar to

the general chemical process of catalysis, inorganic phosphate

was released and the activated iron species were regenerated. It

was observed that the initial velocity of G6P hydrolysis in the

aged iron solution can be described by the Michaelis–Menten

equation, a typical behavior of biocatalysis, in a range from 5 to

250 mM (Fig. 1c, and 1d).

1

no

~ 9:985 | 108 z1:371|109

½G6P�or2 ~ 0:997� �

(2)

where [G6P]o is in M and vo is in M s21.

The maximum initial velocity of G6P hydrolysis was about 1

nM s21, or 3.6 mM h21, and the Michaelis–Menten constant

(Km) was 13.7 mM. This is in strong contrast to previously

reported promotion effects by metals29–32 and minerals.33–37

The promotion effect of G6P hydrolysis can be extended to

2500 mM in this aged iron solution with a k of 6.53 6 1027 s21,

and t1/2 of 295 h. It should be pointed out that the concentration

of phosphorus in the solution was 103–105 higher than that of

iron (e.g., 16.5 nM Fe and 2500 mM G6P).

The measured hydrolysis rate constants of the initial 20 mM

G6P with different sources of iron, made by acid-forced

hydrolysis, are listed in Table 1. There were some differences

in the hydrolysis rate constants among the different salts. The

half-life for 4 month aged Fe(NO3)3 (16.5 nM), FeCl3 (10 nM)

and Fe(ClO4)3 (10 nM) was 37.8, 58.6 and 78.4 h, respectively,

though they were in the same order of magnitude. The nitrate

ion, which is found in ferric nitrate, is also an oxidising agent

though it has been suggested that nitrate exerts little, if any,

effect on the hydrolytic polymerization of ferric ion.52,53 Both

ferric and ferrous salts have a promoting effect, although the

ferrous ions would have been partially oxidized to ferric ion

during the aging process.54

These aged iron solutions also catalyzed the hydrolysis of other

sugar phosphates, including glycerol 2-phosphate (3-carbon, G2P),

ribose-5-phosphate (5-carbon, R5P), and fructose 1-phosphate

(6-carbon, F1P) (Table 2). Preliminary results show that the

hydrolysis rate constants at a given sugar phosphate concentration

are the same order of magnitude among these different sugar

phosphates. It is also noted that the rates of 5-carbon and 3-carbon

sugar phosphates (R5P and G2P) are higher than those of

6-carbon sugar phosphate (G6P and F1P). As expected, the

promotion effect was also found for the other phosphorus ester

compounds, including the energy metabolism compounds (AMP,

ADP and ATP, and even polyphosphate and pyrophosphate) as

well as the RNA model compound (4-nitrophenyl phosphate

ester). However, no promotion effects were observed for the

hydrolysis of phosphonates (C–P bonded compounds, e.g.,

2-aminoethylphosphonic acid, phosphono-formic acid) and inositol

hexakisphosphate (IP6) (data not shown).

Further experiments with a range of 1 to 10 000 nM ferric

standard solution (pH # 6.3) were carried out to investigate the

contribution of iron concentration to the aging process. The

hydrolysis rate constants of initial 20 mM G6P in all these

nanomolar (1 to 1000 nM) aged iron solution increased with

Table 1 Hydrolysis rate constant of 20 mM G6P in aged inorganic iron solutionsa

Fe source Manufacturer Aging time (month) Total Fe conc. (nM) Rate constant (1026 s21) Half-life (h)

Fe(NO3)3 J.T.Baker 14 16.5 30.17 6.4Riedel-de Haen 4 16.5 5.08 37.8

100 2.96 65.66 1000 18.77 10.3

Iron standard solution(metal Fe in 0.3 M HNO3)

J.T.Baker 4 1 2.38 81.02.5 4.85 39.77.5 7.22 26.7

50 6.38 30.2100 6.55 29.4200 6.58 29.2500 8.14 23.6

1000 5.86 32.8FeCl3 J.T.Baker 16 2 6.59 29.2

10 4.95 38.9Riedel-de Haen 4 10 3.28 58.6

100 0.83 231.9Fe(ClO4)3 Aldrich 4 10 2.46 78.4

100 0.61 318.3Fe(NH4)2(SO4)2 EM Science 16 16.5 9.49 20.3Fe-EDTA Self-made 16 16.5 4.24 45.4a The hydrolysis rate constant of initial 20 mM G6P in DIW is 1.53 6 1027 s21, and the half-life is 1255 h.

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aging time at room temperature (22 ¡ 2 uC) (Fig. 2). After

4 months of aging, the hydrolysis reaction rate constant of initial

20 mM G6P in aged iron solutions increased from 0.15 to 10 (61026 s21), and the half-life of G6P hydrolysis decreased from 2

months to y1 day (Fig. 2). It is noted that the time of aging

needed to achieve an appreciable G6P hydrolysis rate constant

was closely related to the total iron concentration. For iron

concentrations less than 100 nM, it took less than a month;

whereas for higher iron concentrations (200 to 1000 nM), it took

more than two months. However, no direct correlations between

the hydrolysis rate constants at the same initial concentration of

G6P and the total iron concentrations were observed, which

might imply that the catalytic characteristics of aged iron

solution is very complex. No significant promotion effect was

observed if the iron concentration was more than 1000 nM, even

if the aging time was extended up to 14 months (data not shown).

3.2. Inhibition effect of tetrahedral oxyanions on the hydrolysis of

G6P

When the tetrahedral oxyanions were introduced into this aged

iron solution, the hydrolysis of G6P was significantly inhibited

(Fig. 3a). In the experiments with initial 20 mM G6P in the 10

month aged 1000 nM iron solution, addition of 10 mM PO4

reduced the hydrolysis reaction rate constant to 60% of the

control (without PO4), and the half-life was 5.51 h. When the

PO4 concentration was increased to 40 mM, the rate constant was

Table 2 Sugar phosphate hydrolysis in aged inorganic iron solutions

Sugar phosphate Aged Fe solution Initial OP (mM) Rate constant (1026 s21) Half-life (h)

Glycerol-2-phosphate (G2P) Fe standard solution, 7.5 nM, 4 mo. 10 12.69 15.220 6.40 30.150 3.45 55.8

500 0.51 374.3Fe(NO3)3, 1000 nM, 6 mo. 20 30.12 6.4FeCl3, 2 nM, 16 mo. 20 5.29 36.4Fe(NH4)2(SO4)2, 16.5 nM, 16 mo. 20 11.44 16.8

Ribose-5-phosphate (R5P) Fe standard solution, 7.5 nM, 4 mo. 10 13.92 13.820 8.15 23.6

Fe(NO3)3, 1000 nM, 6 mo. 20 25.19 7.6FeCl3, 2 nM, 16 mo. 20 7.24 26.6Fe(NH4)2(SO4)2, 16.5 nM, 16 mo. 20 16.16 11.9

Fuctose-1-phosphate (F1P) Fe standard solution, 7.5 nM, 4 mo. 10 8.66 22.220 5.25 36.4

Fe(NO3)3, 1000 nM, 6 mo. 20 17.08 11.3FeCl3, 2 nM, 16 mo. 20 5.29 36.4Fe(NH4)2(SO4)2, 16.5 nM, 16 mo. 20 8.50 22.6

Fig. 2 Hydrolysis rate constant (k) of 20 mM G6P as a function of aging

time in solutions of different iron concentrations.

Fig. 3 Glucose-6-phosphate hydrolysis in a 10 month aged , 1000 nM

Fe(NO3)3 solution at room temperature (22 ¡ 2 uC). (a) Kinetics of

hydrolysis of G6P (20 mM initial) in the aged iron solution with no

addition, and with addition of 1 mM MoO4, 1 mM WO4 and 10mM PO4.

(b) Percentage reduction of the hydrolysis rate constant (k) of initial 20 mM

G6P as a function of the concentration of different tetrahedral oxyanions.

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reduced to 40%, and the half-life was 10.34 h. In the presence of

1 mM MoO4, the hydrolysis rate constant was reduced to 78%,

and the corresponding half-life was 4.01 h. In the presence of

10 mM MoO4, the hydrolysis rate constant was further reduced

to 12%, and the corresponding half-life was 26.3 h. When the

molybdate was increased to millimolar concentrations, no

hydrolysis of G6P was observed even at a higher concentration

of G6P (100 mM). The strongest inhibition effect was observed in

the presence of WO4. The rate constant was approximately 43%

when 1 mM WO4 was added into the aged iron solution; the

corresponding half-life of G6P was 7.42 h. When the concentra-

tion of WO4 was increased to 10 mM WO4, the rate constant was

further reduced to 3%, and the corresponding half-life was

extended to 109.3 h. It was found that the percentage reduction

in hydrolysis reaction rate constant (y) is a rational function of

the tetrahedral oxyanion concentration (x in mM), which can be

expressed as the following equation

y ~100

1 z ax(3)

where the coefficient a is related to the strength of the inhibitor.

In this experiment, a was 1.373, 0.493 and 0.0649 for tungstate,

molybdate and orthophosphate, respectively, with the r2 of fitted

equation in a range of 0.97–0.99 (p , 0.0001) (Fig. 3b).

The inhibition effect was also closely related to the initial

concentration of the G6P (Fig. 4), and the modes of tetrahedral

oxyanions and G6P were competitive. The aged iron solutions

might be bound to either the tetrahedral oxyanions (PO4, MoO4,

and WO4) or G6P to form two different intermediates, but

cannot bind to both at any given moment (Fig. 5). The

intermediates with G6P release IP, and the process of G6P

hydrolysis is completed with the regeneration of the active

binding sites on the aged iron species. The intermediates with the

tetrahedral oxyanions prevent the binding of G6P, and also have

a dissociation reaction to regenerate the active binding sites on

the aged iron species.

Results indicate that the apparent affinity of G6P to the Fe–Fe

bond in the aged iron solution (binding sites) might decrease due to

the competition of the inhibitor (PO4, MoO4 and WO4), while the

maximum velocity of G6P hydrolysis (vmax) was still unchanged.

The catalytic and inhibition behaviors of this aged iron solution in

the presence of 5 to 125 mM G6P can be described by the

Michaelis–Menten equation. From the linear relationship

observed in the Lineweaver–Burk plot, the apparent Michaelis–

Menten constant (Kmapp) can be calculated using the following:

no ~nmax

G6P½ �ozKmapp

G6P½ �o (4)

Kmapp ~ Km 1zI½ �

Ki

� �(5)

Fig. 4 Inhibiting behavior of different tetrahedral oxyanions on the

hydrolysis of glucose-6-phosphate in a 10 month aged, 1000 nM

Fe(NO3)3 solution at room temperature (22 ¡ 2 uC). (a) Effect of initial

concentration of G6P on the initial hydrolysis velocity of G6P.

(b) Lineweaver–Burk plot of aged iron solutions in the absence and

presence of tetrahedral oxyanions.

Fig. 5 Schematic diagram of the catalysis process of G6P hydrolysis in

the presence of the tetrahedral anions in the aged inorganic iron

solutions. E is the aged inorganic iron species; A is the active binding sites

on the aged iron species. S is G6P and I is tetrahedral anions. ES and EI

are the two intermediates.

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where the [G6P]o is the initial concentration of G6P (M), [I] is

the concentration of inhibitor (M), Km is Michaelis–Menten

constant, and Ki is the inhibitor’s dissociation constant.

The Michaelis–Menten equations for G6P hydrolysis in the 10

month aged iron solution were as follows:

without any addition:

1

vo

~ 7:4|108 z2:001|109

G6P½ �or2~0:945� �

(6)

with 1mM MoO4:

1

vo

~ 7:4|108 z1:044|1010

G6P½ �or2~0:998� �

(7)

with 1 mM WO4:

1

vo

~ 7:4|108 z3:42:|1010

G6P½ �or2~0:995� �

(8)

with 5 mM PO4:

1

vo

~ 7:4|108 z8:317|109

G6P½ �or2~0:988� �

(9)

with 10 mM PO4:

1

vo

~ 7:4|108 z1:216|1010

G6P½ �or2~0:997� �

(10)

The Km, the G6P concentration at which the hydrolysis

velocity reached one-half of maximum velocity (vmax/2), was

2.7 mM G6P in this aged iron solution with no inhibitors added,

and the Kmapp with the addition of 1 mM WO4, MoO4, and 5 mM

PO4 and 10 mM PO4 was 46.2, 14.1, 11.1 and 17.1mM G6P,

respectively. Therefore, the Ki of WO4, MoO4, and PO4 was

determined to be 0.06, 0.24 and 1.6–1.9 mM, respectively.

The Km of the aged iron solution was also related to the total

Fe concentration. The higher the concentration of iron in

solution, the lower the concentration of G6P was at one-half

maximum velocity. This probably accounts for the fact that we

could not observe the catalytic effect in the iron solution with

high concentrations (Fig. 2), and the maximum velocities of G6P

hydrolysis in both 16.5 nM and 1000 nM aged Fe solutions were

very similar at around 1 to 1.35 nM s21.

It is interesting to compare the catalytic behavior of aged iron

solution to natural phosphoesterase and their biomimetic,

though the velocities of hydrolysis G6P in aged iron solutions

are much lower than that of phosphoesterase. For natural

phosphoesterase, Km and Ki of PO4 is usually in the millimolar

range;55–60 only Ki of WO4 and MoO4 is in the micromolar

range.57,58,61–63 The value of Km of G6P is 920 mM for phos-

phoesterase extracted from sweet potato50 and 300–310 mM for

those from soybean seed.64 Besides, the modes of molybdate and

tungstate inhibition are noncompetitive.57,58,61–65 Only ortho-

phosphate is competitive57,58 in most cases.

A more significant difference between the aged iron solutions

and the natural phosphoesterase is revealed in their response to

the fluoride ion. While the activity of all known natural

phosphoesterases is very sensitive to fluoride even at micromolar

levels,59,63–69 no change in the catalytic activity of these aged iron

solutions were found even when the final concentration of

fluoride in solutions reached 0.5 M. Another significant

difference is their response to the buffer solutions. Usually, the

natural phosphoesterase still has great activity within the pH

range of the buffer. However, no significant promotion effect on

sugar phosphate hydrolysis was observed when the buffer

solution (e.g., tris, citrate) was introduced into these aged iron

solutions at a similar pH (5.0–7.5) range (ESI Table 1{). The

activity of these aged iron solutions decreased rapidly within

10 min, with the exception of the acetic acid-acetate buffer (the

highest acetate concentration tested was 0.25 M). This is in

agreement with previous observations that many buffers can

significantly decrease the promotion of the metal ion on the

hydrolysis of nucleoside phosphate.29–31 All of these behaviors of

the aged iron solution suggest that they are different from the

natural enzyme.

It is well known that iron speciation changes due to hydrolysis

during the aging process, though controversy still exists over the

identity and stability of the species formed in solution, and of the

interaction of iron with other species.46,70–75 It has been

suggested recently that diiron or polyiron oxide with the oxo-

bridge or hydroxo-bridge (bond) might be formed in the iron

solution during the aging process.74 Based on quantum-chemical

calculations using density-functional theory, dihydroxobridging

binuclear compounds can be present in aqueous solutions, as

binuclear dihydroxobridging [Fe(H2O)4(m-OH)2Fe(H2O)4]n+ and

oxobridging [Fe(H2O)5(m-O)Fe?(H2O)5]n+ (n = 2, 4) cations in

the hydrolysis products of the cations [Fe(H2O)6]m+ (m = 2, 3).76

The hydroxo-bridged Fe–(OH)2–Fe dimers are the structure

units in the polymetric hydroxo complex, which are dependent

on pH and aging time.77,78 Direct experimental evidence has

demonstrated the existence of dinuclear and polynuclear com-

plexes in a 0.1 mM FeCl3 solution. The electrospray ionization

time-of-flight mass spectrometry has detected a variety of

mononuclear and polynuclear iron–oxohydroxo–chloride com-

plexes in Fe solution at a pH range of 2–6.6.79 The (hydr)oxo-

bridged Fe–Fe structure has been confirmed at the interface

of iron oxide (solid) to water.80–83 It was suggested that water

reacts dissociatively on the iron oxide surface, leading to a

structure with both terminal and bridge (hydr)oxy groups. The

m-(hydr)oxo ligand bridge between Fe–Fe also occurs in

nanocrystalline ferrihydrite.84 It is reasonable to assume that

the aged, acid-forced hydrolysed nanomolar iron solutions

comprise species with a m-(hydr)oxo ligand bridge.

The m-(hydr)oxo ligand bridge is a universal feature in

dinuclear phosphoesterase.10,11,15 It has been hypothesized that

a ‘‘phosphoesterase motif’’ provides the scaffold for an active site

dinuclear metal centre in the members of the phosphoesterase

family.12,85–88 They involve the cleavage of phosphoester bonds,

including acid and alkaline phosphatases, bacterial exonucleases,

diadenosine tetraphosphatase, 59-nucleotidase, phosphodiester-

ase, sphingomyelin phosphodiesterase, which is an enzyme

involved in RNA debranching, a phosphatase in the bacterioph-

age genome, as well as the family of ser/thr protein phosphatase

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(PP1, PP2A, and calcineurin).12,15,86 Furthermore, it has also

been reported that the tetrahedral oxyanions inhibit the hydro-

lysis of phosphate ester by these phosphoesterases.59,63,65–69

Moreover, the m-(hydr)oxo ligand bridge was recognized in

artificial phosphatase studies,17–19 which yielded the principles of

binucleating ligand design.24–27 As a result, many biomimetics

with capability to catalyze phosphate ester hydrolysis have been

synthesized based on this special structure.18,21–23,25,28 It is

believed that the catalytic effect of phosphatases and their

biomimetics on the hydrolysis of phosphate monoesters, includ-

ing sugar phosphate, is due to the net transfer of the phosphoryl

group directly to water through a binuclear metal center that

produces inorganic phosphate and that the nucleophile is a

metal-coordinated hydroxide or the bridging hydroxo/oxo

group.10,12,15,51,86,89 Therefore, the common feature between

these aged iron solutions and the phosphoesterases (natural and

synthesized biomimetics complexes) is a kind of acceleration of

electron transfer rate in the structure of the m-(hydr)oxo ligand

between the metals, particularly iron.

Different nanostrutures of iron oxide have been synthesised by

different laboratories,90–95 and it has been shown that they have

different functions.96–99 One example is ferromagnetic nanopar-

ticles with its intrinsic peroxidase activity,96–98 with which it was

demonstrated that their hydrolysis kinetics follow the Michaelis–

Menten equation.96,97 Essentially, similar to phosphoesterase, an

iron center with oxo bonds is also the basic structure of many

peroxidases and their mimetics.100–106 Some PAPs were reported to

have the activity of peroxidases.107,108 Meanwhile, nanoparticles of

iron oxide have been observed in the natural environment,109–112

though some reports mentioned that the nanostructure of iron

oxide can also be mediated by bacteria.113–115 However, as

recognized recently, the structure and reactivity of iron nanopar-

ticles, even in the aqueous solution, changes with time or the aging

process.116 Thus, it is clear that the behavior of iron oxide

nanostructures in nature is very complex. It is believed that

nanometre size iron oxides and (oxyhydr)oxides are ubiquitous in

the earth and range from ultra-fine aerosols to precipitates or

coatings in soil and sediments.110,117–123 Nanomolar levels of ferric

ion might have existed on the ocean surface of the early earth due

to photo-oxidation, even in the absence of oxygen in the

atmosphere,124 though ferrous ion (II) is presumed to be the

dominant form of iron in the sea at that time.125 The elevated CO2

levels that are likely to have prevailed in the atmosphere at that time

could have resulted in a neutral or mildly acidic ocean.125

Moreover, it has been reported that ferric ion is present in the

hydrothermal ecosystem, which is also believed to have been the

realm where life first emerged.126–130 It was also documented that

magnetite (Fe3O4) must have been present in the Earth at that time

through the process of serpentinization, i.e., the reaction of olivine-

and pyroxene-rich rocks with water.131–133

The relevance of this observation is not only to phosphorus

metabolism in biology and the phosphorus cycle in geochem-

istry, but also to the essence of biocatalysts. It demonstrates that

these aged nanomolar inorganic iron(III) solutions perform the

same function as enzymes (natural and biomimetics) that have

metal complexes ligated to either polypeptides or organic ligands.

Natural and biomimetic phosphoesterases and aged inorganic iron

solutions might have the same unique m-(hydr)oxo bridge and

metal center to perform enzymatic functions. This is supported by

the concept of the metal’s role as ‘‘constrained’’ for the selective

uptake and catalytic activity in metallo-enzyme catalysis.134–136

Pauling stated ‘‘the specificity of the physiological activity of

substances is determined by the size and shape of molecules, rather

than primarily by their chemical properties, and that the size and

shape find expression by determining the extent to [which] these

regions of the two molecules are complementary in structure... The

enzyme is closely complementary in structure to the ‘‘activated

complex’’ for the reaction catalyzed by the enzyme’’.137 Therefore,

these aged nanomolar inorganic iron solutions might serve as a

‘‘primitive enzyme’’138 or as an ‘‘inorganic biocatalyst’’. In

other words, all of life’s catalysts are enzymes, although only

protein and RNA (ribozyme) are identified as enzymes in modern

biochemistry.139–144

Conclusions

Aged, acid-forced hydrolysed nanomolar inorganic iron solu-

tions were found to have phosphoesterase activity, which

significantly promoted the hydrolysis of phosphate ester follow-

ing Michaelis–Menten kinetics. Moreover, the catalysis was

inhibited by tetrahedral oxyanions with inhibition strength in an

order of WO4 . MoO4 . PO4. The activity was related to the

aging process and total iron concentrations, but the detailed

mechanism is still unknown. Further work is needed to under-

stand the nature of the (hydr)oxo-bridged Fe–Fe structure in

water and its potential role in organic phosphorus transforma-

tion in geochemistry. However, this observation and reported

intrinsic peroxidase-like activity of ferromagnetic nanoparticles96

demonstrates that ‘‘the chain of life is of necessity a continuous

one, from the mineral at one end to the most complicated

organism at the other’’, as proposed by Leduc.145 The hydro-

lysed iron solutions might be just one of ubiquitous sets of

undiscovered inorganic biocatalysts (enzymes) in nature. Such

inorganic enzymes might act as a bridge between the inorganic

and organic worlds and would have played important roles in the

origin of life. Discovery of further inorganic enzymes might

provide clues on the emergence of life and a potential solution to

the puzzle of ‘‘chicken and egg’’ in life’s evolution.138,146–153

Acknowledgements

X.L.H. greatly appreciates the precious comments of Drs. R.J.P.

Williams, Michael J. Russell, Gerhard Schenk and Jianping Xu

and personal encouragements from Drs. Tsung-Hung Peng,

Peter B. Ortner, Robert Atlas and Mr. Yu-Dong Sun for this

study. X. L. H also thanks Ms. Gail Derr and Dr Raghuraman

Venkatapathy for English editing. Financial support for the

experimental parts of the study was provided by NOAA’s Center

for Sponsored Coastal Ocean Research to J. Z. Z. All

statements, findings, conclusions and recommendations are of

the authors and do not necessarily reflect the views of Pegasus

Technical Services Inc., University of Miami or NOAA or the

U. S. Department of Commerce.

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208 | RSC Adv., 2012, 2, 199–208 This journal is � The Royal Society of Chemistry 2012

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