Phosphoryl transfer from o-carboxyphenyl phosphate to tri(hydroxymethyl)-aminomethane catalysed by alkaline phosphatase from E. coli

PHOSPHORYL TRANSFER FROM o-CARBOXYPHENYL PHOSPHATE TO TRI(HYDROXYMETHYL)AMINOMETHANE CATALYSED BY ALKALINE PHOSPHATASE FROM E. COLZ Departamento

H. A. HERRAEZ, F. J. BURGUILLO,M. G. ROIG and J. L. USERO de Quimica Fisica, Facultad de Ciencias, Universidad de Salamanca, Salamanca, Spain (Received 13 September 1979)

A pH optimum of 8.1 and a weak ionic strength dependence were found in Tris buffer. 2. A light rate enhancement was established with an increase in Tris. 3. Transferase activity to Tris has been tested by ratios measurement of the products at different times and Tris concentrations. 4. Transphosphorylation is not pH-dependent. 5. The release of o-carboxyphenol and Tris-phosphate was Tris-dependent with a maximum at 1.0 M. A decreasing curve was found for phosphate. 6. LineweaverrBurk plot was maintained biphasic in Tris at high ionic strength, opposed to barbital buffer. 7. These results suggest that phosphorylation of the enzyme is not the only controlling step in o-carboxyphenyl phosphate hydrolysis, New effects of Tris are also proposed. Abstract-l.

Stock solutions

INTRODUCTION

from E. coli catalyses both the non-specific hydrolysis of phosphate monoesters and the transfer of phosphate from such esters to acceptor alcohols (Dayan & Wilson, 1964; Wilson et al.. 1964; Neumann, 1969). These transphosphorylation reactions explain the rate increase produced in presence of acceptors such as Tris. ethanolamine, glycerol etc (Dayan & Wilson, 1964; Wilson et al., 1964; Neumann, 1969). Although it has not been shown which the rate limiting step is at alkaline pH (Fernley & Walker, 1966; Halford et al., 1969; Lazdunski & Lazdunski, 1969; Reid & Wilson, 1971a; Halford, 1972; Bloch & Schlesinger. 1973) it appears necessary to assume that the dephosphorylation of a phosphoryl enzyme intermediate represents an important controlling step in the catalytic reaction scheme (Hinberg & Laidler, 1972; Hull et al., 1976). However, it has been reported that o-carboxyphenyl phosphate is a special substrate of which the hydrolysis rate is not increased by acceptor compounds. In this case, phosphorylation is the rate limiting step (Lazdunski & Lazdunski. 1969). In the present paper it is provided data concerning the rate increase of o-carboxyphenyl phosphate hydrolysis by Tris, due to the correspondent reaction of transphosphorylation. According to this, o-carboxyphenyl phosphate shows a similar behaviour to that of the other phosphate monoesters but, in quantity, its hydrolysis would be less catalysed by the enzyme.

Alkaline

phosphatase

MATERIALS

AND

The enzyme was present in suspension (8.X mg/ml in 2.5 M (NH&SO,) and stock solutions were made up using Fife’s method (Fife, 1967): The protein solution was centrifuged at 1600 g for 30 min at 0°C and the portion deposited was diluted in lO.Oml of 0.1 M N-ethylmorpholine butler, pH 8.0, 0. I M NaCI. The enzyme concentration was determined spectrophotometrically at 280 nm, using a molar absorptivity calculated by Schlesinger and Barret (1965). The working samples were prepared immediately before each kinetic run by dilution from the stock solution of i2i-ethylmorpholine and kept at 0_5”C, to elude activity loss in water. Bt#ers

As regards the studies of transphosphorylation, the same nucleophile Tris has been used as the buffer. When a buffer inert to the transphosphorylation was necessary, 5,5-diethylbarbituric acid [barbital] was employed. Buffer’s pH were achieved by adding HCI. pH was determined at room temperature (2%25°C) with a standardized radiometer pH-meter model 5 1, using glass combined electrode type GK 231 I C. The pH was measured before and after each reaction, the variation being always less than 0.05 pH unit. Ionic strengths [I] were adjusted with NaCI. Kinetic procedures A direct and continuous spectrophotometric method for initial rate measurements of o-carboxyphenyl phosphate hydrolysis was carried out at 298 nm with a Schimadzu model QV-50 spectrophotometer to which an automatic recording Servoscribe I-S was adapted. The spectrophotometer was fitted with water-jacketted quartz cuvettes. The temperature was maintained at 25.0 k O.I”C. The initial rates were always measured because a strong inhibition took place due to the products.

METHODS

Reugents

Rates of o-carboxyphenyl phosphate hydrolysis were obtained from the initial slopes of absorbance vs time plots

Chromatographically-purified E. co/i alkaline phosphatase and o-carboxyphenyl phosphate were purchased from Sigma Chemical Co. Tri(hydroxymethyl)-aminomethane and all other chemicals were of analytical reagent grade (Merck).

dc “=___-_I-!!? ld A198 dr AE dt These

511

plots

were linear

he

for %S% of hydrolysis.

(1) Accord-

512

M. A. HFRKAEZ. F. J. BURGL'ILLO.M. G. RCXG and J. L. USERO

ingly. reagent concentration

were calculated in such a way that this “” was achieved in 5-20 min, in this way making it possible to determine accurate Initial overall rates. Transphosphorylation was measured by the following static method: The reaction was stopped at different times by additions of reaction samples (2.0 ml) on 4.0 ml of 2.4 M HCIO,, stored on ice. Subsequently. two 2.0 ml aliquots of the stopped mixture were pipetted into test tubes for the determination of o-carboxyphenoi and phosphate. Inorganic phosphate. determined colorimetricaily by a modification of Allen’s method (Allen. 1940). and o-carboxypheno1 measurements were carried out with a Perkin-Elmer Coleman 55 digital spectrophotometer. Before measuring, analysis samples of o-carboxyphenol were adjusted to pH 9.0 by adding NaOH. In the working pH range (7.0~10.0) only the monoionic form of o-carboxypiIeno1 (pK, = 2.97. pKZ = 13.40) is measured. Its concentration is given by Beer’s law as foilows: (’ z

A 298 ~ <2( _~ c, -
12)

Where cI and Ed are the molar absorptivities of n-carboxyphenol and o-carboxyphenol phosphate at 29X nm, C is the substrate concentration (1.SmM). Molar absorptivities were determined in the common way by absorbance variation measurements as a function of concentration (E, = 3.2X-lO”M-‘cm-‘. cz =0_119~1O”M“cm~‘). Beer’s law test for inorganic phosphate followed a molar absorptivity value of 3.76, IO3 M ’ cm- I Dt 680nm in Allen’s analysis mixtures.

15

r -.-

20

I (MI

Vatues for the slopes and intercepts of all the linear plots (absorbance rs time and o-carboxyphenol concentration cs phosphate concent~dtioil) were calculated using a least squares fit method. RESrLl"S AND

A suitable (Fig.

1Af that

DISCUSSION

experiment of rate against pH shows in Tris buffer there is a ‘*bell-shaped”

profile for the three working ionic strengths (0.1, 0.5 and 1.0 M) with a pH optimum of 8.1. This represents a great difference with regard to p-nitrophenyl phosphate profiles, which are “plateau” type at low ionic strength and keep changing to “bell-shaped” profiles with a pH optimum of 8.5 when ionic strength is increased (Burguillo, 1978). This difference in behaviour suggests the possibility that the side-chain functional groups of the amino acids involved in the enzyme-substrate intermediate are different or, that being the same, their pK would be altered by the binding of o-carboxyphenyl phosphate. Ionic strength dependence on hydrolysis rate remarks (Fig. IB) a weak increase of the rate up to OSM I (optimum); having reached this maximum rate, it decreases at subsequent ionic strength and it becomes stabilized at ionic strength values higher than I.OM. Certainly. this dependence is not as great as in p-nitrophenyl phosphate (Lazdunski & Lazdunski, 1969: Burguillo, 1978), where the rate is triplicated at ionic strength optimum (I .5 M). but it is sufficiently valuable not to be attributable to experimental errors. This light accelerating effect, due to the ionic strength, could be because of small variations in the constants of the acidic dissociation and of the basic

and Tris coneelltra~ion on the initial rate of hydrolysis of o-carboxyphenyl phosphate (VI. catalysed by alkaline phosphatase: A. pH depenFig. I. Effect of pH, ionic strength

dence in the presence of 0.1 M Tris at different ionic strengths [O. 1 M ( x ), 0.5 M (0) and I .O M (0)]. B. influence of the ionic strength in 0..2 M Tris. pH 8. I. C. effect of Tris concentration, at pH 8.1 and ionic strength 1.0 M. Other conditions are: T 25°C. l.SmM o-carboxyphenyl phosphate. 0.3 ;igiml enzyme.

protonation (which increase and decrease respectively with the ionic strength) of the functional groups of the amino acids involved in the catalysis. In this way, the acidic and nucleophilic catalysis, which is assumed (Schwartz, 1963; Lazdunski & Lazdunski. 1966; Katz & Breslow. 1968: Williams & Naylor, 1971) to act in the steps of reaction mechanism, would be favoured on increasing the ionic strength. The aforesaid differences between o-carboxyphenyl phosphate and p-nitrophenyl phosphate with respect to the profiles and amounts of the enlarging effect of the hydrolysis rate, due to the ionic strength, seem to fortify the idea mentioned in the previous section that different groups or the same groups modified take part in the catalysis of each substrate.

E. Co/i alk. phosphatase kinetics Figure 1C shows the increase of hydrolysis rate as a function of Tris concentration, The rate passes through a maximum value at 0.6M Tris. A similar profile was obtained with p-nitrophenyl phosphate but a more displaced optimum (l.OM Tris) was determined (Lazdunski & Lazdunski, 1969; Burguillo, 1978). It has been reported (Lazdunski & Lazdunski, 1969) that the rate of o-carboxyphenyl phosphate hydrolysis is not changed either by ionic strength or by nucleophile reagents at alkaline pH. Thus, it has been concluded that the phosphorylation of the enzyme is the rate limiting step of this particular substrate hydrolysis at alkaline pH (8.0-9.0). These data are opposite to those shown in Figs IS and 1C. Regarding the effect of nucleophile concentration, the probable cause for this discrepancy might be that those authors (Lazdunski & Lazdunski, 1969) have taken in a very extensive range of concentration with few experimental points.

Fig. 2. The pH dependence of the ratio of [o-carboxyphenol] to [phosphate] in the presence of 0.7 M Tris, I 1.0 M. T 25’C, 1.5 mM o-carboxyphenyl phosphate. 4.0 pg/ml enzyme.

Is rhere transphosphorglafion from o-carbo.uypherlyl phosphate (donor) COTris (acceptor), catalysed by alkalirle phosphatase of E. coli? In order to test if the increase effect of rate by Tris could be considered as a result of the transferase activity of enzyme, as occurs with other phosphate monoesters (Dayan & Wilson, 1964; Wilson et al., 1963; Neumann, 1969) the product concentrations at different times have been measured using the static method described in kinetic procedures. The kinetic run was followed until a 60% hydrolysis was achieved. Results for this experiment are given in Table I, where a higher concentration of o-carboxyphenol than phosphate at all times of the reaction is easily noticeable, which clearly indicates lively transphosphorylation [the difference is due to the formation of o-phosphoryl-Tris, (Wilson et al., 1964; Neumann, 1969)]. The ratio of products is time-independent, as was reported for other phosphate monoesters (Barret et al.. 1969). The plot of o-carbqxyphenol concentration vs phosphate concentration is a straight line passing through the origin; the least-squares slope (R) of such Table

Time 1 L I'

I. Product

a line is the most accurate measurement for the determination of product ratio and, therefore, of transferase activity. A value of 1.32 has been found for R, indicating that 0.32 equiv. of o-phosphoryl-Tris has been formed for each equivalent of phosphate. The pH dependence of transphosphorylation

ratios for the hydrolysis of o-carboxyphenyl with Tris as added nu&ophile

o-c<,r boxyphc'm? I??~1

lit",c;i:d?F

to Tris

In order to obtain the pH profile of phosphoryl trransfer from o-carboxyphenyl phosphate to Tris, kinetic runs were followed at various pH in the same manner as in the previous section. Experimental conditions as in Table I. The ratio measurements at different pH have been plotted in Fig. 2. Transferase activity of the enzyme has been found to be pH-independent in the pH range 7.0-9.0 (R = 1.33) decreasing slightly at higher pH (R = 1.28 at pH 10.1). Since the pK of Tris at l.OM ionic strength is 8.35 (Gofdstein, 1976), one may conclude that ionic species of Tris are unnecessary for the transphosphorylation. However, there is a very clear conclusion: o-carboxyphenyl phosphate acts as a donor of transphosphorylation reactions in the whole range of working pH phosphate.

I‘-cclrbi,'KV]>t2< r'CIl,'i‘hi>' pil,dti

'111

I'dtlO

15

0.406

0.306

1.33

75

0.5?9

0.349

1.33

35

0.621

0.452

1.37

55

0.7'36

0 . I,4 'I

1.34

65

0.7al

0.59?

1.37

'75

0.848

0.640

1.33

85

0.893

0.670

1.33

95

o.91a

0.696

1.3:' i'/cr.rq“

1 .33

-+

0.01

M. A.

HEKKAEZ.

F. _I. BURGUILLO.

M. G. ROK; and J. L. USEXO

and Tris (Burguillo, 1978) but, in this case, transphosphorylation is greater (R from p-nitrophenyl phosphate at 1.2M Tris, pH 8.0, I .O M I, is 2.02 (Burguillo, 1978) and from o-carboxyphenyi phosphate at similar conditions is 1.40).

J@ct qf’ Tris concentration ance of reuctim products

0.1

I

05

10 Tris

(Ml

Fig. 3. Ratio of ro-carboxyphetlol] to [phosphate] as a function of Tris concentration, at pH X.I. Other conditions as in Fig. 2.

(7&10.0), which converts this substrate mon phosphate monoester.

into a com-

[o-carboxyphenol]/[phosphate] ratio (R) as a function of Tris concentration between 0.1 and I .2 M has been measured. The kinetic procedure and the experimental conditions were as in kinetic procedures. Results are shown in Fig. 3. There, it is found how R increases linearly as a function of Tris concentration up to 0.7 M, diminishing its slope after this. A similar behaviour is also found with p-nitrophenyl phosphate

on irtitiul rate qf‘ appear-

The transphosphorylation to Tris has been clearly confirmed (Table 1. Fig. 3) as well as the enhancement of the hydrolysis rate in its presence (Fig. 1C); so it seems logical to consider that the hydrolysis activation is due to the transphosphorylation reaction. This has been proved by the following experiment: A solution containing phosphatase (7.5 pg/ml), substrate (2.10-’ Mf and Tris at different concentrations (0.1-1.6 M) was incubate for 5 min at 25°C. To cancel enzyme activity HCIO, was added. Subsequently, aiiquot portions were withdrawn for o-carboxyphenyl and phosphate determinations as it has been described in the experimental procedures. As the hydrolysis percentage achieved in 5 min is less than 3”,;,, the rates so determined are initial rates of the appearance of reaction products. The results are shown in Fig. 4, where the initial rate of formation of Tris-phosphate was calculated bq the difference between the o-carboxyphenol and phosphate rates. Referring to the data. one may conclude -(a) There exists an authentic transphosphorylatjon since the appearance rate of o-carboxyphenol is always greater than that of phosphate. The most simple scheme to explain the hydrolysis. including

1.0

[iris]

(Mf

Fig. 4. Effect of Tris concentration on initial rates of formation of o-carboxyphenol (0). of phosphate (01 and of Tris-phosphate (A). The values given for T&phosphate rates are the difference between o-carboxyphenol and phosphate rates. Conditions: pH X.1, 1 I .OM, T 25 C. 2. IO ~’ M o-carboxyphenyl phosphate. 7.5 i&ml enzyme.

515

E. Coli alk. phosphatase kinetics transphosphorylation.

is:

E+

P,

PI + E

where E is the enzyme, S is the substrate, E’ is the phosphoryl enzyme intermediate, P,, is a phenol, P, is phosphoric acid, P, is Tris-phosphate, and ROH is Tris. This mechanism was reported by Dayan & Wilson (1964). The application of the steady state treatment to this mechanism leads to the following equations: I‘i ZZ kc.,, - EoSo ---.---

Km+-So

k2k; .kc,,, = -- __--_ kz + k; + k,N k,k4N k,,,,, =: -.___~-k2 + k; + k.+N K,

=

&

._

(k;-+k”%

k2 + k; t- k,iv K S = k,’

? !? k,

These k,,, and I(, dependences with regard to nucleophile con~ntration serve to ratify the possible mechanisms of reaction which are proposed for the hydrolysis in the presence of nucleophiles added (N). (b) A slight enhancement is observed in the rate of utilization of substrate (increase in the rate of formation of o-carboxyphenol). This implies that the phosphorylation of the enzyme (k,) is not the only controlling step of the reaction because if it were considered to be so, the hydrolysis rate could not be increased by a transphosphorylation-Tris reaction. To find this hidden reason about which the rate limiting step is at alkaline pH has been the purpose of many studies with other phosphate monoesters and inhibitors (Fernley & Walker, 1966; Halford et at., 1969; Lazdunski & ~zdunski. 1969; Reid & Wilson, 197fa; Halford. 1972; Hinberg & Laidler, 1972; Bloch & Schlesinger. 1973; Hull et al., 1976), but there are various hypothesis. and the conclusions are not clearly defined yet. (c) The profiles (Fig. 4) are curves with a maximum for o-carboxyphenol and Tris phosphate, and a decreasing curve for phosphate. All these coincide with those found for p-nitrophenyl phosphate hydrolysis in our laboratory (Burguillo, 1978), but they differ markedly from the linear behaviour shown by Hinberg & Laidler (1972) for chicken-intestinal alkaline phosphatase, though the probable cause for this discrepancy might be the smaller range of concentration

studied by them. The differences are less with regard to the results established by Dayan & Wilson (1964) for E. coli alkaline phosphatase, even though a different behaviour in the extreme of the range of nucleophile concentration remains. furthermore, our data differs to that reported by Anderson and Nordlie (1967) for the pyrophosphate-glucose transfer reaction, according to which the pyrophosphate hydrolysis rate as well as the phosphate appearance rate are independent from the concentrations of nucleophile (glucose). fd) None of the proposed mechanisms till now (Dayan & Wilson, 1964; Fernley & Walker, 1965; Trentham & Ciutfreund. 1968: Halford et al., 1969; Reid & Wilson. 197la.b; Hinberg & Laidler, 1972; Hull t’t d.. 1976) can explain the transphosphorylation reactions over a wide range of nucleophile concentration since all of them predict hyperbolic or linear dependences for the appearance of the products regarding the nucleophile concentration. but not curve dependences like those in Fig. 4. (e) Since the observed curves for o-carboxyphenol and Tris-phosphate have a maximum about I.0 M Tris, it is logical to believe in the possibility that the nucleophile Tris acts in two different ways, one enhancing the hydrolysis by transphosphorylation and the other, inhibiting it by means of a certain type of reversible inhibition. This hypothesis could explain the inhibition observed in determined concentration of nucleophile, which would be due to a predominance of the inhibitor effect on the activator. ~~77~fi~

,sfud_v ~~o-effrbu.~~~~~~i~~

~~7~sp~7uf~

~?~~~~~~~,~is

Lineweaver-Burk plots have been established in barbital and Tris buffers, and the results obtained have been greatly different (Fig. 5. Table 2). In barbital at high ionic strength (Fig. SA) kinetics are in perfect conformity with the MichaeIis-Menten model whereas, at low ionic strength, the enzyme exhibits substrate activation (Fig. SB). This phenomenon has already been noted by Heppel et a[. (I 962) and Simpson & Vallee (1970) for p-nitrophenyl phosphate. We are in agreement with them in supposing that this substrate activation is, better considered. a homotropit effect of negative cooperativity. The data obtained from the experiments carried out on Tris have been much more interesting. where it was observed that a negative cooperativity does not disappear at high ionic strength (Fig. 5C and 5D, Table 2). Also, this behaviour has been established for p-nitrophenyl phosphate in our laboratory (Burguillo, 1978). The assumption of site heterogeneity reported (Simpson & Vallee, 1970; Applebury et al., 1970) has been reviewed by Bloch and Bickar (1978) who suppose site homogeneity for E. coli alkaline phosphatase. However, this theme is not yet clear. Since a substrate activation remains in Tris at high ionic strength and not in barbital at the same conditions, we suppose that Tris has an allosteric heterotropic effect on the rate of hydrolysis. This is kinetic proof of the existence of a site of allosteric fixation of Tris on the enzyme or on some reaction intermediate. This binding would maintain the interaction between the two active sites necessary for their cooperativity. In agreement with this hypothesis are the Lineweaver-Burk plots at three different Tris concen-

516

M. A.

F. J. BURGCIILLO, M. G. Rorc and J. L.

HI-RRAfz.

USIZRCI

70 60 y ; ;; & m E .G E

50 40 30 20

: z

B l

f

-./’

60

30.

.

D

.

.

> = 50 F(. 0

.

40

.

.

25.

.

20.

.

30 0

10

20

30

40

151

50

0

10

20

30

40

50

I/ [S]w& Fig. 5. Lineweaver-Burk plots for the hydrolysis of o-carboxyphenyl phosphate. In 0.02 M barbital buffers: A. at I.OM ionic strength; B, at 0.035 M ionic strength. In Tris buffers: C. in 0.05 M (0) and 0.5 M (0) Tris at 1.0 M ionic strength: D. in 0.05 M Tris at 0.044 M ionic strength. Other conditions are: In barbital, pH 8.7, 0.1 pg/ml (A) and 0.25 pg/ml (B) enzyme; in Trrs. pH 8.1, 0.1 Icg/ml enzyme (C) and pH 8.0. 0.22 ilg/ml enzyme (D). The temperature was 25’ C and the range of o-carboxyphenyl phosphate concentration 2.10-‘- 2.0 mM.

trations (Fig. 6, Table 3) showing that the negative cooperativity effect disappears at low Tris concentration (0.025 M). With regard to the influence of Tris concentration on the kinetic parameters K, and I; (Table 3). an increase of Vwith Tris concentration is noted, as was hoped. In the terms of K, dependence we do not give conclusions because the rate enhancement by Tris is much smaller for o-carboxyphenyl phosphate than for p-nitrophenyl phosphate. Thus, the experimental uncertainty may be estimable. In the case of p-nitrophenyl phosphate it has been reported (Neumann, Table 2. Michaelis-Menten’s

parameters

1969: Hinberg

v

r

We have found that the rate of appearance of o-carboxyphenol and Tris-phosphate grows with the concentration of Tris in the form of curves with a maximum of about I.OM Tris, and that the rate of phosphate formation decreases slightly also in curve form (Fig. 4). These rates are initial but as the concentration of substrate is very high (2.10e2 M). the said of o-carboxyphenyl

phosphate

k

cat

v

KPl

P,arbitdl

1.0

1.5:‘.10 -1

7.A6

c;a3

0.02"

Rarhltal

0.035

8.69.10-3

,J. ? '3

189

1.09.1c

1.0

7.0A.10-3

5.35

460

1.35.lr,

1.3

2.65.10-?

6.58

566

7.22.10

3.78

315

3.64.1',-2

I" Ti-1s

0.05'" 0.05"

,r1i. Trir.

0.044

5.41.10

-4

hydrolysis

___-.

O.O?b"

0.5

1978) that

1972; Burguillo,

on Tris concentration. SUMMARY

-__c_-

K

& Laidler,,

K, is non-dependent

1.>,1.1'3

-1

-1 -1 -2

7.18

k

cat

643

3.51

3 (12

10.24

i3Al

7.98

636

5.61

Od? -

E. Cali alk. phosphatase kinetics Table 3. Effect of Tris concentration

517

on Michaelis-Menten’s parameters -'?,U?ilC

2.10

0.015

7.78.10

'.I .3 3 .6 __ _

-2

_,

-

1.10

-1

of

Stlbstratc

..,;,:

1.1.10

-7

6.29

5&l

7.78.10

?.63.'$

3.65

311

Y.51?.1'?-d

6,Kl.lC--3

4.07

350

?.07.1u-1

-3

_ _ ____

________..__.~__

....-.__~__._._-._-~-~---~.

rates are authentic maximum rates (v) which, expressed by unit of enzyme, are the catalytic constants (k,,,). Accordingly, we have been analysing the different reaction mechanisms which have been stated for alkaline phosphatase in the presence of nucleophiles (Dayan & Wilson, 1964; Fernley & Walker: 1965; Trentham & Gutfreund, 1968; Halford et ai., 1969; Reid & Wilson, 1971a,b; Hinberg & Laidler, 1972; Hull et al., 1976); thus comparing the dependences which the aforesaid mechanisms predict for k,,, as a function of Tris concentration, with the dependences which are indicated by our data (Figs IC and 4). We have arrived at the conclusion that some of these m~han~sms (Dayan & Wilson, 1964; Hinberg & Laidler. 1972) could explain the observed behaviour up to 0.7 M Tris (which is the range where the dependence appears to be linear), but none of them would explain the experimental profiles over all the range of Tris concentration (Figs lC, 4). As the curve of o-carboxyphenol and Trisphosphate have a maximums we believe that it is reasonable to interpret them in terms of some type of

.

-1

-

2.0

G.24

541

8.77

750

lC.77

1270

.-___-_-_-_I_______

reversible inhibition caused by the nucleophile, without having to believe “a priori” the existence of unknown lateral inhibitions (Wilson 41 ul., 1964). With this idea and based on the results in favour of an altosteric fixation of Tris, it could be supposed that the inhibition caused by the nucleophile. is due to the formation of non reactive complexes with the enzyme or with the reaction intermediates. Henceforth, the most important conclusion of this work is, contrary to what has been believed up to now. that o-carboxyphenyl phosphate behaves the same as the other phosphate monoesters when nucleophiles are added, although the effects are decreased. In agreement with that conclusion, also in absence of nucleophiles added, o-carboxyphenyl phosphate displays a k,,, four times less than k,,, for ordinary phosphate monoesters (Lazdunski & Lazdunski, 1969). We also conclude that o-carboxyphenyl phosphate shows itself to be a very interesting substrate which may lead to a greater understanding of alkaline phosphatase through its quantitative differences regarding the rest of phosphate monoesters. Ackrlowirdgrr,terlrs-We thank the Ministerio de lnvestigaci6n y Universidades for an award to M. G. Roig. Also, we appreciate the help and cooperation of Miss Aidil Diaz Garcia and Mr. Joseph Walshe in preparing the manuscript.

REFERENCES

0

l--___---~. 0

10

20 l/(S](m

30 M“

40

50

1

Fig. 6. Lineweaver-Burk plots for the hydrolysis of o-carboxyphenyl phosphate at three different Tris concentrations, 0.025 M (a), 0.3 M (0) and 0.6 M (0). The range of substrate concentration was 2.10- ‘-2.0 mM. Other conditions are: pH 8. I, I 1.OM, T XC. 0.07 &ml enzyme.

ALLENR. J. L. (1940) The estimation of phosphorus. Bioc&f. J. 34, 858-865. ANDERSON W. B. & NORI>LIER. C. (1967) Inorganic pyrophosphate-glucose phosphotransfcra~ activity associated with alkaline phosphatase of E. co/i. J. hiol. &em. 242, 114-l 19. APPLERURYM. L.. JOHNSON B. P. & COLEMAN J. E. (1970) Phosphate binding to alkaline phosphatase. Metal ion dependence. .I. hiol. them. 245, 496&4976. BARRETH., BUTLERR. & WIL~GNI. B. (1969) Evidence for a phosphoryl-enzyme intermediate in alkaline phosphatase catalyzed reactions. Biochumisrry 8, 1042- 1047. BLOCH W. & SCHLESINGER M. J. (1973) Phosphate content of ~s~~~~ric~iaco& alka&e phosphatase and its effect on stopped-Row kinetic studies. J. hiol. &VU. 248, 57945805.

M. A. HEKRAEZ,

518

F. J. BURG~XLLO, M. G. R~IG and J. L. US~RO

BLOCH

W. & BICKAR D. (1978) Phosphate binding to ~.s~~~~,rj~~i[ueofi alkaline phosphatase. Evidence for site homogeneity. J. hictl. &51. 253. 621 I-62 17. Bt.;a S. E. (1972) Escherichiu coii alkaline phosphatase. Relaxation spectra of figand binding. ~j~~~~rn. J. 126, 727 -73x. HEI’PEI. L. A.. HARKNESS D. & HII.MOE R. (1962) Substrate specificity and other properties of the alkaline phosphatase of Esch~richia co/i. J. hjol. chrm. 237, 841-846. HrNtn:ac; I. & LAII~LLR K. J. (1972) Steady-state kinetics of enqme reactions in the presence of added nucleophiles. The kinetics of reactions catalyzed bq alkaline phosphatase: the effects of added nucfcophiles. Cojr. 1. Biochw~.

50, I?34 W~ILI

1368.

W. E.. HAL~ORII S. E., G~I,TFKFL’NII H. & SYKES B. D. (1976) Phosphorus 31 N.M.R. study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at afkaflne pH. ~j5~~~~~~7isfr~, 15, 1547.-1561. KA.T% I. & BR~SLOM/ R. (1968) Relative reactivities of

p-nitrophenyf phosphate and phosphorothioate toward alkaline phospllat~ise and in aqueous hydrolysis. J. .4,~r. &In. sot. 90, 737& 7377. LAZIX%SRI C. & LAZUC~~~SKI M. (1966)Etude cinttiquc du mCcanismc d’actlon catalytique de la phosphatase afcafine d’E.vc/trric/tirr co/i. Biochc,n~. hiop/~l~ 3<,r(l f 13. 551 566. LA~IX~SKI C. & LAZIXJNSLI M. (1969) Zn’+ and c‘oL +alkaline phosphatases of E,s&r&ti co/i. Eur. ./. Hioc+/n. 7, 294 -300. NJ-{IMANN H. (I 96Y) Phosphoryl transfer from S-substltutcd monoesters of phosphorothioic acid to various acceptors catalyzed by ~,s~~i~,r~c/li~ co/i. &r. .I. ~j~~~~~~‘/~~. 8. I&- 173. Rrtit T. W. & WILSON I. H. (1971~) Confbrmationaf isomers of alkaline phosphatase in the mechanism of hydrolysis. Biochcwrisrr~~ 10. .%0-3X7. R~.II> T. W. & WILSON I. B. (f97lh) 7’hr E/I:JITI~,,\. 3rd edn. Vol. 4. pp. 373~-115 (Edited by B(1Yt.H P. D.). Academic Press. New York. SCHLLSINGER M. J. & BARRET K. (I 965) Reversible dissociation of the alkaline phosphatase of Escht~richin CO/I. I. Formation and reactivation of subunits. J. hicll. &nr. 240, 478P42Y?. SCHWARTZ J. H. (1963) Phosphor~lation of alkaline phosphatase. Proc. hiar. Acad. &i. G.S.A. 49, 871-878. SIMPSON R. T. & VALLEE B. L. 11970) Negative homotrooic interactions in binding of substrate toBfkafine phosphatase of E.scltc~riclria co/i. Biockmisrr~~ 9, 953 9%. TRCNTHAM D. R. & GC;TFRt:LINI) H. (1968) The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from E,schrrichia co/i. Biochem. J. 106.

455-460. WILLIAMS A. & NAYL~R R. A. (1971) Evidence for S,? (P) mechanism in the phosphoryfat~on of alkaline phosphatase by substrates. J. chrnz. SIX. (B), 1973-1979. WILSON I. B., DAYAN J. & CYR C. (lY64) Properties OF alkaline phosphatase from Esclrerichiu colitransphosphoryfation. J. hi& rketn. 239, 4182~4185.