Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 1, October 1, pp. 32’7-337, 1981

Human Placental Alkaline Phosphatase: Effects on Conformation Ligands Which Alter Catalytic Activity’ JAMES

B. ORENBERG,

JOHN

M. SCHAFFERT,2

AND

HOWARD

by

H. SUSSMAN*v3

Department of Chemistry, San Francisco State University, San Francisco, Califonzia 9.&W, and the *Laboratory of Experimental Oncology, Department of Pathology, Staqfcrrd University School of Medicine, Staqford, Cal$?n-nia 9.@05 Received

March

30,198l

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 f 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 * 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pKaPP of 10.29. The remaining 32 f 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 M guanidine-hydrochloride normalizes all 3’7 + 1 tyrosine residues (PK.,, = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), L-phenylalanine (uncompetitive inhibitor), D-phenylalanine (noninhibitor). and M$+ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with L-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mgz+ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.

Alkaline phosphatase (EC 3.1.3.1) is a class of phosphomonoesterases that cati This work was supported by a grant from San Francisco State University Faculty Development Fund, NIH-Biomedical Sciences Support Grant Program, NIH 5-SO5 RR07120, Dean’s Fellowship, Stanford University School of Medicine, and Public Health Service Grants HD04562 from the National Institutes of Child Health and Human Development (J.B.O. and J.M.S.) and CA13533 from the National Cancer Institute (H.H.S.). ’ Present address: Varian Associates, Palo Alto, Calif. 94304. 3 Address for reprints and correspondence: H. H. Sussman, Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, Calif. 94305.

alyzes phosphohydrolytic and phosphoryl transfer reactions with little specificity for either substrate or phosphoryl acceptor. Considerable information is known about the alkaline phosphatase of Escherichia coli as a result of studies investigating its chemical and physical properties, mechanism of action, and genetic control of synthesis (1). Mammalian phosphatases have not been as well studied (2, 3). Of these, the placental alkaline phosphatase (PAP)4 is the best characterized and the physical ’ Abbreviations used: phosphatase; CD, circular ene glycol.

327

PAP, placental dichroic; PEG,

alkaline polyethyl-

0903-9361/81/110327-11$02.00/0 Copyright All rights

0 19fil by Academic Press, Inc. of reproduction in any form reserved.

323

ORENBERG,

SCHAFFERT,

properties and kinetic behavior of this enzyme, which is a glycoprotein, have been reported elsewhere (4-6). There is considerable interest in PAP because it is synthesized by some neoplasms in which it is not phenotypically expressed (“ecto*pic synthesis”) (7), and has become useful as a marker in studies investigating regulation of gene expression in eukaryotes (8, 9). The differential inhibition in response to pH, certain amino acids, polypeptides, and metal ions has been used to distinguish PAP from other alkaline phosphatase isoenzymes (2, 3). The basis for this distinction is not well understood. In this investigation, we have employed uv difference spectroscopy to examine the state of the 36-38 tyrosyl residues per enzyme dimer by observing the ionization behavior of these phenolic hydroxyl groups from pH 7.0 through 12.6. The results provide information about surface and interior tyrosine residues (10-12) which indicate a change in molecular conformation of the enzyme during alkaline titration. We have also investigated the effects on enzyme conformation of ligands which alter catalytic activity of PAP using the techniques of solvent perturbation difference spectroscopy and circular dichroism. The ligands used were inorganic phosphate (competitive inhibitor), L-phenyialanine (uncompetitive inhibitor), D-phenylalanine (noninhibitor), and Mg2+ ion (activator). MATERIALS

AND

METHODS

Reagents. High purity guanidine-HCI with low absorbance in the ultraviolet range was purchased from Schwarz-Mann (Orangeburg, N. Y.) and was used without further purification. Tris buffer was prepared from Sigma (St. Louis, MO.) reagent grade Trizma-HCl. 2-Mercaptoethanol was Sigma Type 1 grade. Monoethanolamine, monoethylamine, ethylene glycol, and glycerol were Baker (Phillipsburg, N. J.) analyzed reagent quality; polyethylene glycol 300 (A!& 235-315) was Baker grade. Reagent-quality N-acetyl-btyrosine ethyl ester (AtyrE) and N-acetyll-tryptophan ethyl ester (AtrpE) were from Fox Chemical Company (Los Angeles, Calif.). The amino acids, L-phenylalanine and D-phenylalanine, were “A” grade from Calbiochem (La Jolla, Calif.). All other reagents and solvents used were of the purest commercial quality available and were used without

AND

SUSSMAN

further purification. All solutions were prepared with doubly glass-distilled water. Human placental alkaline phosphatase was isolated and purified according to previously described procedures (13). Amino acid analysis of the purified enzyme gave a value of 3’7 -C 1 tyrosine residues per dimer (14). Tryptophan content was measured using the procedure of Edelhoch (15) and was determined to be 10 * 1 per dimer. Protein concentration was determined by measuring the absorbance at 2’78 nm using a molar absorptivity of 9.05 X 10’ (4). Enzyme activity was measured using gnitrophenyl phosphate as substrate following the procedures of Bessey et al. (16). All calculations used a value for the molecular weight of the enzyme dimer of 116,006 (4). Spectrophotmetric observations. Spectral observations were made on a Cary Model 15 double-beam recording spectrophotometer and a Gilford Model 240 single-beam spectrophotometer. For direct absorbance measurements, standard l-cm semimicro silica cells were employed (Precision Cells, Inc., Hicksville, N. Y.); for difference absorbance measurements, split compartment mixing cells for difference spectroscopy (Pyrocell Manufacturing Co., Westwood, N. J.) and Type 56 divided cell with stoppers (Precision Cells, Inc.) were used. In difference measurements, the Cary 15 was operated using the expanded scale slidewire (0.1 absorbance full scale) and servo slit. By proper adjustment of either or both sensitivity and dynode voltage, the slit was never permitted to exceed 1.0 mm. A baseline was recorded for every difference spectral run with the split compartment cells in place containing the exact concentration of reagents used for the difference spectrum (1’7). Circular dichroic measurements recorded in net molar ellipticities (degrees cmr/dmol) were obtained on a JASCO J-40 automatic recording spectropolarimeter with a dedicated Nova Model S-40 computer. Recordings of circular dichroic spectra were taken in the 200- to 340-nm region. The protein concentrations used for the pH difference studies were in a range of 2.0-10.0 X 10e6 rd. The protein solutions were all 0.1 M in NaCl to maintain a constant ionic strength at p = 0.1. All pH measurements were made on a Corning Model 12B or an Orion Model 301 pH meter using a Beckman Model 39037 ceramic junction combination electrode. The spectrophotometric titration of phenolic hydroxyl groups was effected by measuring the difference in absorbance between alkaline and neutral solutions of enzyme at each of two wavelengths (295 and 244 nm). The alkaline solutions for the forward titrations were produced by adding the requisite amount of sample enzyme to a mixture of Tris buffer and 0.1 M NaOH for pH 7.0-9.5, ethanolamine for pH 9.510.5, monoethyiamine for pH .1&5-11.7, and 0.1 M NaOH beyond pH 11.7 (buffer concentration in all cases was 0.02~ in the final enzyme solution). For the reverse titration the sample enzyme solution was

EFFECTS I

OF LIGANDS I

1

I

ON HUMAN I

I

I

PAP + MEOH

PLACENTAL

ALKALINE

1

TABLE APPARENT

3 ‘-

329

PHOSPHATASE I

NUMBEROFEXPOSEDTYROSINE

RESIDUES

BY SOLVENT

PERTURBATION

DIFFERENCESPECTROSCOPY

System’ PAP PAP PAP PAP PAP

3-

(native) + D-phe (0.015 M) + L-phe (0.15 M) + P, (0.050 MI + MgZf (0.30 mu)

‘Range

280 290 WAVELENGTH, FIG.

spectra volume

300 “m

1. Theoretical and actual molar for the perturbation of PAP methanol (MeOH).

uv difference with 20% by

either dialyzed against the appropriate pH solution of 0.1 M NaCl plus buffer or volumetrically titrated toward neutrality with pH 7.0 Tris buffer. Enzyme solutions in 4 M guanidine-hydrochloride with 0.01 M 2-mercaptoethanol were allowed to stand for 4 h at 25°C before titration. For solvent perturbation difference measurements, protein concentrations ranged from 4 to 7 X 10m6 M. The pH of the enzyme solutions was maintained at 7.0 k 0.2 by the use of 0.01 M Tris-HCl buffer. The pH of solvent perturbants (methanol, ethylene glycol, polyethylene glycol) was adjusted to 7.0 -+ 0.1 with standard HCl or NaOH. The pH of all solutions was monitored before and just after the completion of a solvent perturbation measurement and was observed to be constant. Solutions used for circular dichroic measurements were prepared in the same way as those used in the solvent perturbation difference spectral measurements.

Methanol

Ethylene glycol

18.9 k 0.8 16.1 14.5 12.8 20.7

19.8 * 1.2 18.0 21.9 14.3 22.6

of PAP concentrations

was 4.0 -

PEG 10.5 * 1.2 11.7 9.1 8.7 11.5

7.0 X 10e6 1.

of the procedures may be found in the references (2325). Twenty-seven wavelength points in the 285- to 295-nm region of the spectrum were used in Herskovits’ curve-fitting procedure, where a theoretical curve based on tyrosine and tryptophan model data was fitted to the solvent perturbation difference spectrum (21, 22). The distances in nanometers that the theoretical difference spectrum was red-shifted along the wavelength axis to give the best curve fit were found to be 1.0 nm for methanol, 1.5 nm for ethylene glycol, and 1.0 nm for polyethylene glycol. Figure 1 shows the computer-generated best fit curve for the perturbation of PAP with methanol. The relative uncertainty in average chromophore exposure values obtained from solvent perturbation experiments is calculated to be f 7% (21) The standard deviations of tyrosine exposure values reported in Table I ( +0.8-1.2 residues) were about 4-11% of the average exposure values. Relative standard deviations in tryptophan exposure values (Table II) ranged from about 10 to 40% ( +0.2- + 0.9 residues). The greater uncertainty of tryptophan exposure is a result of the low degree of exposure of this chromophore in the enzyme. Since tryptophan exposure values are low and contain large relative uncertainties in their calculation, small changes in the degree of exposure of

Evaluation of data from spectrophoknnetric methods. From the pH difference experiments, the number of tyrosyls ionizing and the pKapp of these groups were determined using a graphical plot of [H+] Ah’ vs AE in the manner of Tachibana and Murachi (18). The slope of a line from a computer generated leastsquares fit yielded an apparent pK value, pKpPP For calculating the number of tyrosines ionizing in a pH range, the value of 2.33 X l@ was used for A,!&, and 1.10 X ld for aEW, AE being the difference molar absorptivity for the ionization of a single tyrosyl group (10, 19, 20). The apparent number of exposed tyrosines and tryptophans from solvent perturbation difference spectroscopy was calculated using procedures introduced by Herskovits (21, 22). A detailed explanation

TABLE APPARENT

NUMBER

II

OF EXPOSED

TRYPTOPHAN

RESIDUESBYSOLVENTPERTIJRBATION DIFFERENCESPECTROSCOPY Ethylene System’ PAP PAP PAP PAP PAP

(native) + D-phe (0.015 m) + L-phe (0.015 M) + Pi (0.050 M) + I@+ (0.32 mtd)

‘Range

of PAP concentrations

Methanol 2.4 + 0.9 1.1 1.0 0.8 1.8

PEG

glYCO1

1.5 + 0.2 1.1 1.5 0.3 2.4

wa8 4.0 + 7.0 X lo-’

0.7 k 0.2 0.9 0.2 0.2 0.8 Y.

ORENBERG,

330

SCHAFFERT,

295nm

Wavelength,

nm

FIG. 2. Difference spectra of human alkaline phosphatase (2 + 10 X 10e6 M) at several pH values. The pH of the reference enzyme solution in the difference cuvet was 7.00 + 0.01. The spectra were recorded after allowing steady-state ionization to occur.

the tryptophans of PAP as shown in Table I are not significant. The circular dichroic spectra of native PAP and PAP with additives were obtained in the 200- to 240nm range (far uv) and the 260- to 300-nm range (near uv). Contributions to the molar ellipticities due to the added ligands (which are high for D- and L-phenylalanine) were cancelled in the recorded spectra by the use of appropriate reference solutions. The relative uncertainty in the far ultraviolet CD measurements was determined from four replicate CD measurements for the native enzyme. Relative standard deviations of + 8% for the 200- to 223-nm negative band and f 22% for the 208-nm negative band were calculated. The relative uncertainties of the near ultraviolet CD bands were also within this range (S-22%). Four CD determinations were run in the far uv on another protein, bovine serum albumin, in the presence of 0.15 M D-phenylalanine. The results gave relative standard deviations less than the above values for the 208- to 223-nm CD bands. It was assumed, therefore, that the optically active stereo isomers of phenylalanine used in this study did not increase the uncertainty in CD measurements. RESULTS AND DISCUSSION

pH Dgference

Spectral Studies

AND SUSSMAN

pronounced. This peak is attributable to the ionized form of the tyrosine chromophore. A corresponding absorption spectrum was observed at 244 nm, which is a second maximum for the ionization of tyrosine. In the present study 5 of the total 37 f 1 tyrosine residues in the PAP dimer were found to titrate normally between neutrality and pH 10.14 (Fig. 3). Titration of these residues exhibited no time dependence of ionization, and these can be considered to be “free” and in the exposed state (“free” meaning “free-to-ionize”). The close correspondence of a theoretical titration curve for 5 tyrosines (Fig. 3) and the experimental curve lends proof that only 5 of the 3’7 tyrosine residues are freely accessible to solvent in the pH range between 7.00 and 10.14. The change in difference absorbance, L4, at 295 nm as a function of time was evaluated in the pH range from 10.11 through 12.27. The time dependence of the titration of these tyrosine residues is illustrated in Fig. 4. Below pH 10.5 the difference absorbance at 295 nm reached its maximum value instantaneously after adjusting a neutral enzyme solution to that pH and remained constant for several hours. At pH values greater than 10.5, a time-dependent ionization was observed

n ,060 cc a Q 050

9 g = E z ‘6

Expenmentol

-

,030

.020

.o IO

I 8

9

IO

II

I

I

I2

I3

PH

Ultraviolet difference spectra of PAP at pH 9.25, 10.40, and 11.10 are shown in Fig. 2. As the pH of the sample enzyme was increased, the characteristic difference absorption peak at 295 nm became more

FIG. 3. Comparison of the actual alkaline spectrophotometric titration curve of human placental alkaline phosphatase (9.3 X lo-’ M) at 295 nm with a theoretical curve for five tyrosyl residues ionizing with a pKaPP = 10.29.

EFFECTS

OF LIGANDS

I

ON HUMAN

I

I

PLACENTAL

ALKALINE

I

I

PHOSPHATASE A

h

I”

331

I”

,c

.22-

,

pti a-9

12.27

-

Y

.I6 -

pH "2

-

f pH Il.57 J

fA-

r/ pH -c -t

5

IO

IS

20

Ih,h,I\,

25

30

"

4

--c

pHll.lI

-

-a pH 10.50 -0 z pH IO.7

--c + 0

II.34

-

60 "

120"

-

160

MINUTES

FIG. 4. Time dependence of the difference absorbance of human (6.6 x 10-z M) at 295 nm in the pH range of 10.11 - 12.27.

as indicated by the gradual increase in AAB5 (Fig. 4). At these elevated pH values a steady state for the ionization of tyrosine groups was approached after 3 h. The time-dependent ionization indicates that buried tyrosyl residues are becoming exposed to the alkaline solvent and contribute to the measured difference absorbance. The necessity of having to titrate groups which maintain molecular structure in order to allow the molecule to unfold and expose the interior tyrosines for ionization would also account for the observed pK,,, of 11.7 for these “buried” tyrosine residues being higher than was measured for the 5 “free” tyrosines (pK,,, = 10.29) and for the “free” tyrosines of the unfolded enzyme in 4 M guanidine-hydrochloride (pKapp = 10.08). The forward alkaline titrations of PAP in 0.1 M NaCl and in 4 M guanidine-HCl are shown in Fig. 5 with a theoretical curve constructed for 36 tyrosine residues. In the titration in 0.1 M NaCl, the steadystate difference absorbance at higher pH values was approached after 3 h (Fig. 4), while in guanidine-HCl the equilibrium difference absorbance occurred instantaneously throughout the pH range. If the data of the forward alkaline titration are replotted according to the method of Tachibana and Murachi (18), the pK,,, for ty-

placental

alkaline

phosphatase

rosine ionization in any desired pH range may be determined. An example of this type of treatment is shown in Fig. 6 for the titration of PAP in 4 M guanidine-HCl (pH range 9.86-11.72). In addition, the number of tyrosine residues ionizing may be calculated. The results of the Tachibana and Murachi plots for enzyme titrations in 0.1 M NaCl and in 4 M guanidine-HCl in different pH ranges are shown in Table

I

I

I

I

I

0

I

FIG. 5. Spectrophotometric titration curves of human placental alkaline phosphatase (9.3 X lo-’ M) at 295 nm in 4 M guanidine-HCl and in 0.1 M sodium chloride and a theoretical curve for 36 tyrosine residues. The pH of the reference enzyme solution in the difference cuvet was 7.00 -+ 0.01. Each spectral pH point was recorded after attainment of an ionization steady state.

332

ORENBERG.

SCHAFFERT.

A=295nm

pH=966-11772 4M Gumdins

AE

-HCI

x IO-3

FIG. 6. Tachibana and Murachi method of evaluation of difference titration curves for the alkaline titration of PAP in 4 M guanidine-HCl at 295 nm in the pH range 9.86 - 11.72 where AE is the difference molar absorptivity.

III. In the low pH region (8.93 -+ 10.14), the results of the two difference peaks of tyrosine (295 and 244 nm) are in agreement. The amino acid composition of the enzyme has been reported to contain 14 residues of cysteine as cysteic acid (5). The close correspondence of the data for ionization of free tyrosine residues in the low pH range at 295 and 244 nm indicates then that none of the reported 14 cysteine residues is free to ionize in the pH range and does not add to the difference absorbance at 244 nm. The data of Table III also indicate that all of the tyrosine residues in the PAP dimer are successfully titrated under highly alkaline conditions (pH range 10X12.63). Thirty-eight tyrosine residues of the enzyme were observed to be titrated in 0.1 M NaCl and this is in close agreement with the 36 residues obtained for the enzyme mOlWUle after titration in 4 M guanidine-HCl and also agrees with the 37 f 1 tyrosines obtained from amino acid analysis by chromatography (5,6,26). The close correspondence of the curve for the forward alkaline titration of PAP in 4 M guanidine-HCl with a theoretical titration curve constructed for 36 tyrosines ionizing with a pKaPP of 10.08 (Fig. 5) and the observed instantaneous ionization of all tyrosine residues indicate that all of the tyrosines are exposed in this concentration

AND

SUSSMAN

of guanidine. Since the enzyme is completely dissociated into monomer subunits in guanidine (4), the exposure and titration of all tyrosines of the enzyme in 0.1 M NaCl at high pH may similarly be an indication that monomers are the predominant species. The experimental titration curve for the enzyme in 4 M guanidine-HCl departed from the theoretical curve (Fig. 5) at very high pH values. This was caused by turbidity in the titration solution. Reverse titrations from high pH to neutrality were conducted with enzyme solutions that had been adjusted to pH 9.85 and 11.27 (Fig. 7). These titrations were performed by the addition of volumetric amounts of 2.0 M Tris buffer at pH 7.0; each recorded point represents the steadystate value. When the enzyme was adjusted to pH 9.85, and then back-titrated to neutrality, the back-titration curve corresponded exactly to the forward titration curve within experimental error. This indicates that below pH 10 the free, exposed tyrosines are reversibly titrated, as would be expected for “free” tyrosyl residues in proteins. It also indicates that up to this pH there is no change in molecular structure that allows titration of buried tyrosines. However, when the reverse titration was conducted with enzyme raised to the high alkaline pH of 11.27, the reverse titration curve was displaced above the forward titration curve (Fig. 7). The displacement indicates that the number of TABLE

III

CALCULATED pKapp VALUESANDNUMBEROF TYROSINE RESIDUES IONIZING IN THE ALKALINE TITRATIONOFPAPACCORDINGTOTHE METHODOFTACHIBANAANDMURACHI No. of Solvent” 0.1 M NaCl 0.1 M NaCl

0.1 M N&l 4 M GuanidineHCI

'Range of PAP

pH range

-

8.93 8.93 10.81 9.86 -

eoneentrations

10.14 10.14 12.63 11.72

P&P

10.23 10.23 11.7 10.08 was 20 -

tyrosine residues ionizing

A bm)

5.1

295

5.4 38 36

295 295

10.0 X 10.’ II.

244

EFFECTS

OF LIGANDS

ON HUMAN

PLACENTAL

.OS8 2 4 -

.06-

: x

PH

FIG. 7. Comparison of forward and reverse titration curves of PAP (9.3 X low6 M) at 295 nm. Reverse titrations were commenced after 3 h exposure of the enzyme to the appropriate initial pH. Open circles represent reverse titration of PAP from an initial pH of 9.85; solid circles represent reverse titration of PAP from an initial pH of 11.27. The solid line is the forward titration curve of PAP in 0.1 M NaCl from pH 7.0.

tyrosines ionizing is increased, thus giving a higher difference absorbance value, and suggests an irreversible alteration in molecular structure with internal groups coming to the enzyme surface. Under similar experimental conditions, a loss in antigenicity and a reduction in specific activity of the enzyme has been observed (6).

Solvent Perturbation Dichroism Studies

and Circular

Native enzyme. Exposure values for tyrosine and tryptophan in native PAP were obtained from solvent perturbation difference spectra using methanol, ethylene glycol, and polyethylene glycol (PEG) as solvent perturbants and Herskovits’ method of evaluation (21, 22). The results for all perturbants are given in Tables I and II. With ethylene glycol, about 54% (20 out of 37) of the tyrosines of PAP appear exposed in the native enzyme and about 15% (1.5 out of 10) of the tryptophans. Laskowski (27) has stated that in general around 50% of the chromophores in protein are exposed to the solvent en-

ALKALINE

PHOSPHATASE

333

vironment. The exposure of tyrosines of PAP using methanol as perturbant is similar to that obtained using ethylene glycol, and both of these perturbants give exposure close to the expected value of 50%. The lower exposure obtained with PEG is related to the “size effect” of this perturbant. The small value for the percentage of tryptophans exposed is similar to the result by Donovan (28) for aldolase: 1 out of 10 tryptophans, while 14 out of 42 tyrosines were exposed. The relation of perturbant size to the apparent exposure of tyrosine and tryptophan residues of PAP can be interpreted in terms of the existence of one or more crevices in the native enzyme. The exposure of tyrosines and tryptophans to PEG (9.2 A mean molecular diameter) is about half the exposure obtained with ethylene glycol and methanol (4.3 and 2.8 A, respective mean molecular diameters). This large drop in exposure reveals that some of these chromophores are accessible only to the smaller-diameter perturbants and are assumed to be housed in one or more clefts or crevices in the enzyme. Since access is possible for ethylene glycol but not possible for PEG, it can be assumed that the diameter of the crevice opening is between 4.3 and 9.2 A. Tryptophans of native PAP did not show a small increase in exposure due to the increase in range effect5 of the perturbant. Instead, a large relative drop in tryptophan exposure was observed as the perturbant size was increased from 2.8 A (methanol) to 4.3 A (ethylene glycol). Although this behavior again suggests that crevices (2.8-4.3 A diameter) exist, it cannot be safely postulated because the change in exposure of tryptophans is of the same magnitude as the uncertainty in exposure for methanol perturbation. The environment of the tyrosine side chains was also elucidated from the sign, magnitude, and position of some of the bands in the near uv (>250 nm) circular dichroic (CD) spectrum of the native en’ Range effect: The relative turbant to perturb partially

(27).

ability of a solvent perexposed chromophores

334

ORENBERG,

SCHAFFERT,

AND

a polar environment, i.e., aqueous solvent. The 283-nm band is also attributable to tyrosine, and the position of this negative band is solvent dependent (29). This band may occur between 282 and 289 nm; the closer it is to 282 nm, the more polar is the environment of the tyrosine chromophores (29). The position of the 283-nm band (Fig. 8A, C) also indicates that more than half of the tyrosines of PAP are in a polar environment. Since this band appeared as a shoulder, its position could

zyme (Fig. 8A, C). The spectrum displays negative bands at 276 and 283 nm and positive bands at 290 and 298 nm. The 290and 29%nm bands are attributable to the tryptophan side chain (29). The 276-nm band is attributable to tyrosine side chains; this band is negative in polar solvents or environments and positive in nonpolar solvents or environments (29). The fact that this band is negative in the CD spectrum of the native enzyme indicates that the tyrosines of PAP, on an average, reside in I

I

1 I

1 I

,

I

so-

SUSSMAN

I 1 A-

I

/

I

I

I

I

I B-

ZO60-

-6

O-

-2o-

I I , ,-v--d ‘\ b PAP. PO

-so-

nm

c-2 5; -7J 100 x

,

z is F a _I d

-4o“6 z OE 0.2

I 260

I

I / I 260 300 WAVELENGTH,

c G

I

I 320

1

i i ?I 5

,

I

SO-

/

I

I

I

I

I ‘t,,/PnP+L-ph* I I I I

i 60-

40-

-

I c

_

2

-10 oI

I

I I I I I 200 220 240 WAVELENGTH, nm I

r

I

I

I

I

/ D _

2.0I

i

PAP+O-phe -lO.O-

-4o-

I 260

260 300 WAVELENGTH,

320 nm

I I I I 200 220 WAVELENGTH,

I 240 nm

I

FIG. 8. (A) Near ultraviolet (uv) circular dichroic (CD) spectra of PAP, PAP + Pi, and PAP + M& (B) far uv CD spectra of PAP, PAP + Pipand PAP + MgZ+; (C) near uv CD spectra of PAP, PAP + D-phe, and PAP + L-phe; (D) far uv CD spectra of PAP, PAP + D-phe, and PAP + L-phe.

EFFECTS

OF LIGANDS

ON HUMAN

PLACENTAL

only be estimated. The near uv circular dichroic spectral results correlate with the solvent perturbation results of Tables I and II, which show that more than half of the tyrosine chromophores are accessible to methanol and ethylene glycol perturbants. The far ultraviolet CD spectrum of PAP at pH 7.0 (Fig. 8B, D) was used as a reference spectrum for the native state of the backbone or secondary structure of the enzyme. Negative bands occurred at 208 and 222 nm. Changes in these bands in the presence of added ligands were considered evidence of induced changes in the secondary structure of the enzyme (29). Native enzyme plus inorganic phosphate. In the solvent perturbation studies the addition of inorganic phosphate (Pi), a competitive inhibitor of PAP as 0.050 M Na2HP04, resulted in large reductions in the numbers of tyrosines and tryptophans exposed to t.he perturbants (Tables I and II). Using methanol, the perturbant with the smallest mean molecular diameter (2.8 A), exposure of tyrosines decreased by 6 residues and exposure of tryptophans decreased by 1.6 residues from the native values. A similar reduction in the exposure of these chromophores was observed with ethylene glycol (4.3 A mean molecular diameter) as perturbant. Exposure of these chromophores was not significantly decreased from the values for native enzyme with PEG (9.2 A mean molecular diameter) as perturbant. Since the smaller perturbants are more sensitive to detecting changes in the crevices of the enzyme, it would appear that a large part of the effect of Pi on enzyme structure occurs at this level; i.e., the partial closing or blocking of the crevices due to an induced conformational modification. In the near uv CD spectrum, a small redshift in the CD band at 276 nm (Fig. 8A) and a lower negative value in the presence of 0.05 M Na2HP0., were observed compared to the native enzyme. These findings indicate a less polar character of the environment of the tyrosine side chains; i.e., less exposure cif these residues to the aqueous solvent. The 283-nm CD band of the native enzyme was shifted to about 285 nm in the presence of Pi; this also indicates

ALKALINE

PHOSPHATASE

335

a less polar environment for the tyrosine side chains. In the far uv CD spectrum, the addition of Pi to the enzyme caused large alterations relative to the spectrum of the native enzyme (Fig. 8B). The increase in magnitude of the negative CD bands shown in Fig. 8B suggests that a conformational change has occurred in the backbone structure of the enzyme (29). Native enzyme plus D- and t-phenylalanine. L-Phenylalanine is an inhibitor and D-phenylalanine is a noninhibitor of PAP catalytic activity (30). In solvent perturbation studies with 0.015 M D-phenylalanine, the exposure of tyrosine and tryptophan residues to methanol (Tables I and II) was observed to be slightly lower than with native PAP. Changes in the exposure of these chromophores to ethylene glycol and PEG, however, were so close to the uncertainties for the exposure values of native PAP that they were judged to be without significance. In the CD studies with D-phenylalanine, the near uv CD spectrum of PAP (Fig. 8C) showed slight changes in the 2’76- and 282to 289-nm negative bands which is interpreted as a small decrease in the polar environment of the tyrosines. The far uv CD spectrum of PAP in the presence of Dphenylalanine (Fig. 8D) was within the experimental error (standard deviation) of the CD spectrum of native PAP, indicating that little or no conformational change had occurred in the secondary structure (backbone) of the enzyme. Solvent perturbation studies in the presence of 0.015 M L-phenylalanine showed a significant decrease in the exposure of tyrosine and tryptophan residues of PAP to methanol. The magnitude of the change was significantly more with L- than with D-phenylalanine (Tables I and II). With ethylene glycol and PEG as perturbants, the decrease in exposure of these residues was close enough to the native values to be attributable to experimental uncertainty. These observations suggest that the change in exposure is occurring mainly at the topographical or crevice level; i.e., the addition of L-phenylalanine induces a conformational change which causes the partial closing or blocking of some of the

336

ORENBERG,

SCHAFFERT,

crevices of the enzyme accessible only to methanol. In the near uv CD spectrum (Fig. 8C), the 2’76-nm CD band was smaller with L-phenylalanine than in the native case or with D-phenylalanine, and the position of the 283-nm band was again shifted to about 285 nm. Both of these facts indicate a decrease in environmental polarity for tyrosines, and therefore, a decrease in exposure of the tyrosines. The far uv CD spectrum of PAP plus L-phenylalanine (Fig. 8D) displayed small but significant increases in the magnitudes of the 208- and 220- to 223-nm peaks, indicating that a change in the backbone structure of the enzyme had occurred. Native enzyme plus i&f+. The solvent perturbation spectrum with 0.32 InM MgClz as additive indicated a small but significant increase in exposure of the chromophores of PAP with all perturbants (Tables I and II), except for exposure of tryptophan residues with methanol as perturbant. The increase in exposure was greater than the experimental uncertainty. The near uv CD studies with Mgz corroborated these results; the 278-nm negative band was of larger magnitude than with the native enzyme (Fig. 8A), indicating more exposure of tyrosine residues. The position of the 282- to 289-nm band was approximately the same (283 nm) as in the CD spectrum for the native enzyme. The far uv CD spectrum of the Mg2+-enzyme system (Fig. 8B) was within experimental uncertainty of that for the native enzyme and was interpreted to indicate that no measurable change in the secondary structure of the enzyme had occurred upon the addition of Mgz+. CONCLUSIONS

The ability to reversibly titrate the five free tyrosines between pH 7.0 and 10.14 suggests a stable enzyme conformation exists in this pH region. The persistence of an increased number of ionizing tyrosines (>five) after reverse titration from pH 11.27 to pH 9.28 most probably indicates that the molecule remains unfolded after exposure to high alkaline pH and does not achieve the conformation of the native dimer. This explanation is consis-

AND

SUSSMAN

tent with the conclusion of sedimentation equilibrium studies of the pH-dependent monomer-dimer association reactions (6). The failure to reform to the conformation of the native enzyme after exposure to high pH may be a result of the enzyme being a glycoprotein with the sugar groups affecting the refolding of the peptide chains. Spectrophotometric evidence for other globular proteins in aqueous solution indicates that many of the phenolic side chains of tyrosine are buried in the interior of the molecule (31). These groups are inaccessible to water and are unable to ionize in the pH range where ionization would normally be expected to occur. The presence of these residues in the interior of the molecule is consistent with the existence of hydrophobic regions which are thought to account for the compact, symmetrical shape of globular proteins (31). PAP is a globular protein and the findings in the present investigation suggest that the interpeptide bonds of the native enzyme dimer may be stabilized by hydrophobic interactions. The observation that most of the tyrosines in PAP are resistant to titration until the molecule is raised to a pH where the native dimer undergoes irreversible unfolding is supportive of these being interior residues which participate in stabilizing the conformation of the native molecule. A possible mechanism for the major change in comformation above pH 10.5 is that while internal tyrosines in a protonated state are stable in a hydrophobic environment, when ionized to tyrosyl anions, charge repulsion would contribute to disruption of the hydrophobic bonding. Ultracentrifugation studies which demonstrated the molecule is stable in high ionic strength had suggested that the dimer structure is maintained by hydrophobic interactions (6), as has been reported for other proteins (31). The finding of a major change in conformation in the region of pH 10.5 is of interest because the pH optimum for catalytic activity of PAP is in the same region, pH 10.5-11.5 (32). Conformational changes with pH have been proposed by Lazdunski and co-workers in a model explaining the catalytic mechanism of calf

EFFECTS

OF LIGANDS

ON HUMAN

PLACENTAL

intestinal alkaline phosphatase (33), which also is a glycoprotein dimer of similar or identical subunits (20). The solvent perturbation and CD studies showing changes in the conformation of PAP induced by inorganic phosphate, a competitive inhibitor, give insight into the interaction of this ligand with the enzyme at neutral pH. The existence of a phosphorylated enzyme intermediate (EP) in the mechanistic scheme for catalysis is generally accepted (2). It has also been postulated that a conformational change results from substrate binding and that this distortion provides the driving force for the reaction (2). The spectral observations of this study are in accord with this postulate and indicate that the binding of Pi induces a conformational change in the enzyme which could alter the active site and thereby decrease enzyme catalytic function. The solvent perturbation studies with L-phenylalanine which demonstrated the topographic changes of partial closing or blocking of crevices on the enzyme are consistent with the mechanistic postulate that L-phenylalanine acts as an uncompetitive inhibitor preventing the dissociation of the phosphoryl enzyme intermediate (34). The activation of alkaline phosphatase by Mgz+ has been postulated to occur because of an increase in molecular diameter of the enzyme (2). The data showing increased exposure of chromophores in the solvent perturbation studies but no change in the uv CD spectrum are consistent with an increase in molecular diameter without a change in backbone or secondary structure of the enzyme.

6. SUSSMAN,

H.

B&him. 7. GREENE,

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H. H. (1973)

Nat. Acad. Sci. USA 70, 2936-2940. 8. CHOU, J. Y., AND ROBINSON, Phgsiol. 92.221-232.

J. C. (1977)

J. Cell

9. HAMILTON, T. A., TIN, A. W., AND SUSSMAN, H. (1979) Proc. Nat. Acad. Sci. USA 76,

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323-

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13. SUSSMAN, H. H., SMALL, P. A., JR., AND COTLOVE, E. (1968) J. Biol Chem. 243, 160-166. 14. MOORE, S., AND STEIN, W. H. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. VI, pp. 819-831, Academic Press, New York. 15. EDELHOCH, H. (1967) Biochemistry 6,1948-1954. 16. BESSEY, 0. A., LOWRY, 0. H., AND BROCK, M. H. (1946) J. Biol. Chem. 164, 321-329. 17. ERICKSON, J. O., AND BRAMSTON-COOK, R. (1973) Varian Znstrum. Appl. 7 (Part 4) 10-12. 18. TACHIBANA, A., AND MURACHI, T. (1966) Bio-

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chemistry 7, 2533-2542. 23. OHNISHI, M. (1971) J. Biochem. 69.181-189. 24. OHNISHI,

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REFERENCES 1. REID, T. B., AND WILSON, I. B. (1971) in The Enzymes (Boyer, P. D., ed.), Vol. IV, pp. 373-415, Academic Press, New York. 2. FERNLEY, H. N. (19’71) in The Enzymes (Boyer, P. D., ed.), Vol. IV, pp. 41’7-447, Academic Press, New York. 3. FISHMAN, W. H. (1974) Amer. J. Med. 56,617-650. 4. GOTIZIEB, A. J., AND SUSSMAN, H. H. (1968) Biochim Biophys. Acta 160.167-171. 5. HARKNESS, D. R. (1968) Arch. Biochem. Biophys. 126.503-512.

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