ARCHIVES
OF
BIOCHEMISTRY
Vanadium
AND
BIOPHYSICS
ion Inhibition V. LOPEZ, of Chemistry,
Department
175, 3138 (1976)
of Alkaline Phosphatase-Catalyzed Ester Hydrolysis1 T. STEVENS, San
Francisco
AND
State
Phosphate
R. N. LINDQUIST
University,
San Francisco,
California
94132
Received October 20, 1975 OxovanadiumW) ion (V02+) and vanadium(V) ion (VO,-) are potent competitive inhibitors of the Escherichiu co2i alkaline phosphatase-catalyzed hydrolysis ofp-nitrophenyl phosphate. The dissociation constants (KJ of the enzyme-inhibitor complexes have been determined as a function of pH over the pH range 7.0-10.0. Vanadium(V) & zz 2.2 x 10m6M, pH 8.0, Tris buffer) binds about as well as inorganic phosphate and has a pH-Ki profile similar to that of phosphate. OxovanadiumUV) ion binds about one to two orders of magnitude better than phosphate over this pH range (K, = 4 X, lo-’ M, pH 8.0, barbital buffer). Theorell-Yonetani plots show that vanadium(V) binds mutually exclusively with inorganic phosphate, and VO*+ also appears to bind in this manner at lower inhibitor concentrations. Tris buffer lowers the effectiveness of VO*+ as an inhibitor by a factor of four to five. These observations indicate that VOI- can bind quite specifically at the phosphate-binding site on the enzyme. The more potent inhibition by V02+ suggests that the ensyme-complexed inhibitor may have some resemblance to the metastable intermediate formed during the hydrolysis of phosphate esters.
Oxovanadium(IV) and vanadium(V) ions complexed with uridine are potent’inhibitors of ribonuclease and bind greater than three orders of magnitude more tightly than the corresponding substrate molecule uridine 2’,3’-phosphate (1). The ability of vanadium to form complexes of a trigonal bipyramidal nature led to the proposal that the vanadium complexes were acting as transition state analogs by virtue of their possible structural resemblance to the substrate portion of the transition state for ribonuclease-catalyzed hydrolysis of uridine 2’,3’-phosphate (1). Enzymatic phosphor-y1 transfer reactions in general are considered to proceed through a transition state involving a trigonal bipyramidal pentacovalent species in which entering and leaving groups occupy apical positions (2). If oxovanadium ions or their enzyme-chelated forms can be shown to be analogs of such metastable intermediates, then V02+ and VO,- ions have a wide potential in structural and mechanistic investigations of enzyme* This
work
was
catalyzed phosphate ester hydrolysis reactions, which are numerous in biological systems. Oxovanadium(IV) has excellent electron spin magnetic resonance properties, and Chasteen and co-workers (3, 4) have recently used V02+ to probe the active site of carboxypeptidase and carbonic anhydrase by replacing the zinc ion in the native enzymes. While our studies were in progress Van Etten et al. (5) reported that V02+ and other transition metal ions are excellent reversible competitive inhibitors of acid phosphatase. This enzyme hydrolyzes phosphate esters via a covalent phosphoryl-enzyme intermediate where an Nphosphorylhistidine (enzyme) is thought to be involved. Alkaline phosphatase (EC 3.1.3.1) is a metalloprotein with a broad substrate specificity, which catalyzes the hydrolysis
supported by NIH Grant No. GM
20817 to R. N. L. 31 Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
32
LOPEZ, STEVENS
AND LINDQUIST
phosphate, 25°C) monitored at 400 nm is about 25-30 of a number of phosphate esters presump,mol of substrate hydrolyzed per minute per milliably via a phosphoryl-enzyme intermediate where an active-site serine residue is gram of protein in agreement with Simpson and phosphorylated (6). The transition states Vallee (15). The final preparations were stored in for the formation and decomposition of this 0.01 M Tris at pH 8.0 at 4°C. Enzyme activity decreased only slightly over a period of 6 months. phosphoryl intermediate should resemble Disodium p-nitrophenyl phosphate was pura trigonal bipyramidal pentacovalent spe- chased from Sigma Chemical Company. Stock solucies in which the entering and leaving tions of oxovanadium(IV) were made by dissolving groups are axial (2, 7). There are few po- Fisher reagent grade VOSO,.2H,O in 0.01 M HCl. tent competitive inhibitors of alkaline The V(IV) concentration was determined by titraphosphatase aside from inorganic phos- tion of the stock solution with KMnO, (16) and was approximately 80% of the value expected on the phate and arsenate (6, 8). Recent studies by Ohlsson and Wilson (9) have shown basis of the weight and the empirical formula. Stock that periodate is the most potent reversi- solutions of V(V) were prepared by dissolving Fisher-purified NH,VO, in hot water; immediately ble competitive inhibitor of this enzyme upon cooling the pH was adjusted to 8.0 with NaOH. yet discovered (Ki = 0.5 x 10m7M, pH 8.6). All other chemicals were reagent grade and were This ion, however, binds at a different site obtained commercially. All solutions were prepared than phosphate and has a different pH-Ki from glass-distilled deionized water. Since V(IV) is profile than for phosphate or for substrate oxidized by molecular oxygen to V(V) at pH values binding. greater than about 4 (17, la), all solutions used in Oxovanadium(IV) ion (V02+) is known experiments with V(IV) were prepared from O,-free to form five-coordinate complexes with wa- water, and purified nitrogen or argon was passed ter and other ligands (10, 11). It is possible through them. The solutions were transferred using pipets and syringes that had been flushed with nithat V02+ could rapidly and reversibly form a chelated complex with the enzyme trogen. which resembles in some manner the tri- Kinetic Measurements gonal bipyramidal transition state in the The initial rates of hydrolysis of p-nitrophenyl alkaline phosphatase-catalyzed hydrolysis phosphate were measured spectrophotometrically of phosphate esters. It is also possible that using a thermostated (25°C) Gilford 240 spectrophovanadium(V) might adopt a trigonal bi- tometer with a Radiometer REC 51 recorder and a pyramidal structure since crystalline hy- 0.0-0.10 full scale absorbance mode. The reaction contained 2.8 ml of buffer and 200 pl comdrated metavanadates (VO,- * H,O) are solution prised of substrate, water, and inhibitor. The reacfive-coordinate with oxygen atoms, and tion was initiated by the addition of 10 ~1 of stock the geometry is approximately trigonal bi- enzyme solution to the cuvette and monitored at pyramidal (12). This paper describes the 400 nm for several minutes. Alternatively, in reacpotent inhibition of Escherichia coli alkations where inhibitors were present, the substrate line phosphatase by oxovanadium(IV) and was added last to initiate the reaction. Typically, an absorbance of 0.01 to 0.03 was needed to determine vanadium(V) ions. EXPERIMENTAL
PROCEDURES
Materials The enzyme was prepared from E. coli by the osmotic shock technique of Neu and Heppel (13) and purified by chromatography on DEAE-cellulose using a sodium chloride gradient (14). Alternatively, E. coli alkaline phosphatase chromatographically and electrophoretically purified was purchased from Worthington Biochemicals (BAPF). This preparation had comparable activity to that isolated and gave similar K, and KS values in the different buffers used. Enzymatic activity of such preparations (pH 8.0, 1.0 M Tris?, 1 x 1O-3 up-nitrophenyl 2 The abbreviations used are: PNPP, p-nitrophenyl phosphate; Tris, tris(hydroxymethyl)aminomethane; DEAE-, diethylaminoethyl.
the initial slopes. By monitoring the first few percent or less of the reaction, substrate hydrolysis and inorganic phosphate inhibition were kept to a minimum. The pH of the reaction mixture was measured at the end of each kinetic run. The initial velocity values were determined using the extinction coefficients determined for the p-nitrophenolate anion in the two buffers used. The reaction solutions were diluted 1:l with 0.1 N NaOH after completion and the A, values thus obtained (e400 = 1.84 x lo4 M-I cm-‘, 0.02 M Tris, 0.5 M KCl; l d0,,= 1.97 X 104 M-l cm-‘, 0.02 M barbital, 0.5 KCl). The presence of V02+ or VO,- in the solution did not alter these extinction coefficients, The Henderson-Hasselbalch equation was used to determine the percentage ofpnitrophenolate species present at each pH value assayed, and the observed change in absorbance was corrected accordingly. In addition, rates were nor-
VANADIUM
INHIBITION
OF ALKALINE
malized from day to day to a standard value of enzymatic activity which was checked at the beginning and the end of each set of kinetic measurements. The protein concentrations of the stock enzyme solutions were determined spectrophotometritally according to EjB. (278 nm) = 7.2 (19). Special precautions were taken to exclude oxygen in all assays involving VO*+. The cuvette was initially flushed with purified nitrogen, and the buffer, enzyme, and VO*+ solutions were added under a flow of nitrogen. After flushing the solution with nitrogen for a minute, the cuvette was stoppered and equilibrated in the thermostated spectrophotometer compartment for several minutes, and then substrate solution was quickly added to initiate the reaction. The stock solutions were all flushed with nitrogen afier every few runs and kept stoppered. No detectable enzyme inhibition was found with sulfate ion at concentrations up to 1O-3 M Na,SO,. Values of K, for p-nitrophenyl phosphate were determined at several pH values (25°C) in 0.02 M Tris-HCl and 0.02 M ethanolamine and 0.02 M barbital, all in 0.5 M KCl. In addition, the Ki for phosphate was determined at two different substrate concentrations (1 x 10m4and 5 x 10m5M) using six concentrations of P, (1 x 1O-5-7.5 x 10m5M) in 0.02 M TrisHCl, 0.5 M KC1 buffer, pH 8.0 and 25°C. The enzyme concentration was 0.43 pg/ml. At each substrate concentration the quotient of the uninhibited and inhibited rate (V,,/V) was plotted against the inhibitor concentration. These plots gave straight lines intersecting at 1.0 as expected for a competitive inhibitor. The Ki value determined from these plots (Ki = 1.5 x 1Om6Ml is in excellent agreement with a similar determination by Snyder and Wilson (K, = 1.3 2 0.1 x 10m6M) (20). All intercepts and slopes were determined using an unweighted least-squares program.
I I O7
PHOSPHATASE
I 9
I 9
IJ IO O
PH
FIG. 1. Enzyme activity as a function of pH in 0.02 M barbital, 0.5 M KC1 buffer (Ml and K, as a function of pH in 0.02 M Tris, 0.5 M KC1 (O), 0.02 M ethanolamine, 0.5 M KC1 (01, and 0.02 M barbital, 0.5 M KC1 (0). The r’action mixtures contained 1 x lo+ to 5 x 10m6M ofp-nitrophenyl phosphate in a total volume of 3.0 ml with an enzyme concentration of 0.57 or 0.31 pglml (barbital buffer). Specific activities were measured at a substrate concentration of 1 x 10m3 M. Initial rates were calculated from the initial slopes of AA/At at 400 nm, 25”C, using the calculated E values and appropriate product ionization factors (see Experimental). Values of Km were calculated from plots of l/initial velocity vs l/[Sl.
obtained at pH 9.0 in both Tris and ethanolamine btier systems. RESULTS A plot ofK, vs pH (Fig. 1) shows that K, values increase as the pH is increased Effect of pH on PNPP Hydrolysis from 7 to 10, consistent with other pubThe enzymatic activity and substrate and inhibitor binding ability of E. coli al- lished data (8, 21). Strong product inhibition is an inherent difficulty in this K, kaline phosphatase are sensitive functions so that only approximate of the buffer and ionic strength of the reac- determination values are obtained (20). Three buffer systion medium (6,9, 20), and buffers such as tems were used (0.02 M buffer in 0.5 M Tris can markedly affect V,,,. The specific activity of the E. coli alkaline phos- KCl) to cover the pH range 7-10. It appears that the K,,, values do not change phatase-catalyzed hydrolysis of p-nitrophenyl phosphate was measured as a fimc- appreciably as a function of buffer at the used. The K,,, values obtion of pH over the range 7.00-10.00 in 0.02 concentrations M barbital, 0.5 M KC1 at 25°C (Fig. 1). A tained are comparable to previously retypical activity profile results with this ported values (20). buffer and a similar profile was obtained with Tris and ethanolamine buffers in the Inhibition of PNPP Hydrolysis by VanapH range 7-10 which was consistent with dium(V) previously reported values under similar Figure 2 presents kinetic data for the conditions (20, 21). Identical activity was inhibition of E. coli alkaline phosphatase-
34
LOPEZ,
STEVENS
AND LINDQUIST
determined from the slopes of the lines. As with the K, determinations, it appears that the Ki values determined for VO,- do not change appreciably in going from Tris to barbital buffer. At pH 8.0, Ki = 2.2 x lo+ M in 0.02 M barbital, 0.5 M KC1 (vs I& I I I = 2.9 X 1o-6 M in 0.02 M Tris, 0.5 M KCl). The values of Ki forVO,- as a function of pH are plotted in Fig. 3. The Ki between pH 8 and 9 is from 2.5 to 3.5 x lop6 M, a value in the same range as inorganic phosphate under similar conditions (6,20). The inhibition is independent of the length of time (at least up to 30 min) that the inhibitor is preincubated with the enzyme before the addition of substrate. In Fig. 4 the Theorell-Yonetani plot (22) p0Jo3'11 IO-% of reciprocal velocity vs phosphate concenFIG. 2. Inhibition of the alkaline phosphatasecatalyzed hydrolysis of PNPP by VOI- at pH 8.0 and tration in the presence of a fixed concen25°C; PNPP concentrations: 1 x lo+ M (O), 5 x 10-S tration of VO,- and PNPP gives a series of lines over a wide range of VO,M (W, 1 x lo-* M (O), and 2 x lo+ M (0). The parallel reaction mixtures contained 0.02 M Tris, 0.5 M KCI, concentrations (3-100 PM). Parallel lines and enzyme at a concentration of 0.5’7 rig/ml in a to- in a plot of this type indicate that the two tal volume of 3.0 ml. The quotient of the uninhibited inhibitors, Pi and VOS-, bind in a muand inhibited initial rates (VJV) is plotted against tually exclusive fashion. If the lines were the vanadium(V) concentration. All rate values not parallel, it would indicate cooperative, have been normalized to one concentration of en- antagonistic, or independent binding dezyme, and the initial velocities were calculated from pending upon where the lines intersected. absorbance changes of approximately 0.02 to 0.03 At the concentrations of NH,VO, used, esafXer addition of substrate to the preincubated reacsentially all of the vanadium(V) is present tion solution. as the VO,- species and not as a trimer or catalyzed hydrolysis of PNPP by VO,- at higher polymeric species (1). The Ki value pH 8.0. At each PNPP concentration the for VO,- calculated from these lines is very close to that calculated from the V,,/V plots quotient of the uninhibited and inhibited (Fig. 2). rate (V,,/V> gives a straight line intersectAlthough the VdV vs VO,- plots (Fig. 2) ing the ordinate at 1.0 when plotted were linear over a wide range of VO,against the inhibitor concentration. The concentrations, above an inhibitor conceninhibition appears to be linearly competitration of about 1.5 x lop4 M there was a tive over a wide range of VO,- concentradeviation from linearity as the plots tions, and the kinetics can be described by curved downward from the original slopes. the equation This could be a result of polymer formation V by vanadium(V) to a trimer. It has been K, + [PNPP] o=calculated that, in 1.5 x 10m4 M NH,VO, v %a(1 + vo,-/K,) + [PNPP] ’ solutions, about 95% of the V(V) is in the which can be simplified to give (20) monomer state. At increased concentrations more trimer will form, thus decreasl/Ki = [l + (PNPPIK,)] X slope, ing the effectiveness of the inhibition per where Ki is the dissociation constant for mole of VOB- present. the enzyme-inhibitor complex. Inhibition by VO,- appears to be competitive at all Inhibition of PNPP Hydrolysis by Oxovanadium(IV) pH values between 7 and 10. Two to four Initial studies of the inhibition of alkadifferent substrate concentrations were used at each pH. The values of Ki were line phosphatase hydrolysis of PNPP by
:
VANADIUM
I 8
1n10-1 7’
INHIBITION
I
9
OF ALKALINE
I I
IO
PH
FIG. 3. Semilogarithmic plots of K{ vs pH for the inhibition of alkaline phosphatase-catalyzed hydrolysis of PNPP by vanadium(V) (VOJ (D) in 0.02 M Tris, 0.5 M KC1 (pH 7-S) or 0.02 M ethanolamine, 0.5 M KC1 (pH 9.5-10.0) and for the inhibition by oxovanadium(IV) (VO*+) (0) in 0.02 M barbital, 0.5 M KC1 (pH 7.0-9.5) or in 0.02 M ethanolamine, 0.5 M KC1 (pH 10.0). For VO,- inhibition, conditions were as described in the legend to Fig. 1 except that inhibitors were added to give the following concentrations: PNPP, 1 x 10m4-5 x 1O-6 M; VOs-, 1.7 x 10vs-6.7 x 1Om6M; enzyme, 0.57 pg/ml. Kinetic runs using four different substrate concentrations were done at pH 7, 8,9, and 10 and two substrate concentrations for the other pH values. The results at each pH were plotted as in Fig. 2. The& values which are the dissociation constants of the enzyme-inhibitor complex were obtained as described in the text, and an average value at each pH is plotted. For VO*+ inhibition the reaction concentrations were: PNPP, 2.5 X 10m5 M; VO*+,
1.2
x
1O-6-2.4
x
PHOSPHATASE
35
determined in the same manner as described for VOB-. The value determined at pH 8.0 from three kinetic runs was 1.6 x 10e6 M. Thus V02+ is a potent competitive inhibitor of alkaline phosphatase. It binds about twice as tightly as VOB- under the same conditions and has a Ki equivalent to that determined for phosphate (1.5 x lo-+ M). In order to determine if there were any buffer effects on the V02+ inhibition the Ki was determined at pH 8.0 in 0.02 M barbital, 0.5 M KCl. The points for V02+ at pH 8 in Fig. 3 demonstrate that V02+ is a markedly better inhibitor in the barbital buffer system (Ki = 4 x lo-’ M) than in the Tris buffer (1.6 x 1O-6 M). It has recently been reported (9) that Tris buffer has a protective effect toward the inactivation of alkaline phosphatase by permanganate ion. It is possible that this same phenomenon accounts for the reduced binding of V02+ in the presence of Tris, although such an effect is not apparent with VO,- inhibition.
1Om5 M; enzyme,
0.31 pg/ml. The K< for V02+ inhibition at pH 8.0 in 0.02 M Tris, 0.5 M KC1 (0) is an average value from three runs at different substrate concentrations. The V02+ studies and KS determinations were done as above for VO,- inhibition. 000
VO*+ were done in 0.02 M Tris, 0.5 M KC1 ‘buffer at pH 8.0. Kinetic measurements at several different substrate concentrations yielded straight lines intersecting near 1.0 on the VdV axis (Fig. 5), indicating competitive inhibition. A similar study at pH 10 in ethanolamine buffer exhibited a linear relationship also (Fig. 5). The KG was
plosRmTg.lO-%l
4. Theorell-Yonetani plot of initial velocity versus phosphate concentration at varying concentrations of VO,-: no VO,- CO),3 phi CO),5 PM (Ml, 33 PM (Cl), and 100 PM (A), and fixed PNPP concentration (5 x 1Om6Ml, pH 8.0, 25”C, 0.02 M Tris, 0.5 M KCl. Reaction conditions were as described in the legend to Fig. 1 with an enzyme concentration of FIG.
0.31 /&g/ml.
36
LOPEZ,
STEVENS
AND LINDQUIST
the enzyme in Tris or barbital buffer. The parallel lines in the Theorell-Yonetani plot (Fig. 4) indicate that VO,- and phosphate bind to the enzyme in a mutually exclusive fashion. Prolonged preincubation of VO,- with the enzyme does not yield any progressive increase in inhibition, indicating that no oxidation of the enzyme is taking place. Since VO,- exists almost exclusively as the monomeric species in solution at the concentrations used in the inhibition studies, the above results suggest that it may be acting as a phosphate analog in binding very specifically to the site(s) on the enzyme which normally ligands the phosphate anion. In addition, the pH-K, profile for VO,inhibition (Fig. 3) shows a maximum binding of this ion near pH 8.5 and a marked decrease in binding at pH 7 and 10. A similar bell-shaped pH-Ki plot is exhibited of alkaline phosphaFIG. 5. Inhibition of alkaline phosphatase by (8) for the inhibition VO*+ in 0.02 M Tris, 0.5 M KCl, pH 8.0, PNPP, 2 x tase by inorganic phosphate at 25°C with a 10e5 M (O), and 5 x 1O-5 M (W) and in 0.02 Methanolminimum at pH 8.0-8.5. Phosphate inhibi30
I-
1
1
amine, 0.5 M KCl, pH 10.0, PNPP, 5 x 10e5 M (0). Reaction conditions were as described in the legend to Fig. 2.
1
Another possible explanation is that Tris forms a complex with VOZ+ in the concentration range studied and that barbital either does not complex or forms a much weaker complex. Kinetic plots for the inhibition of E. coli alkaline phosphatase by V02+ in the pH range 7.0 to 9.5 in barbital buffer (Fig. 6) exhibit a linear dependence on V02+ in the concentration range used. The lines intersect the VdV axis near 1.0 suggesting a competitive type inhibition. The Ki values in this pH range were calculated as previously described and are plotted in Fig. 3. DISCUSSION
Vanadium(V) ion (VO,-) and oxovanadium(IV) ion (V02+) are both potent inhibitors of E. coli alkaline phosphatase. The former ion clearly exhibits competitive inhibition with the phosphate ion over the pH range 7.0-10.0 (Figs 1 and 2) and binds about as well as inorganic phosphate ion at pH 8.0 (Ki = 2.2 x lop6 M for VO,- vs Ki = 1.5 x 1O-6 M for phosphate). In contrast to V02+, the VO,- ion binds equally well to
FIG. 6. Inhibition of alkaline phosphatase by VOz+ at pH 8.0 (W, pH 7.0 (01, and pH 9.5 (0) (least-squares line for pH 9.5 has a steeper slope than line for pH 7.0). The reaction mixtures contained a PNPP concentration of 2.5 x 10m5M, 0.02 M barbital, 0.5 M KCl, and enzyme at a concentration of 0.91 pg/ml. All reactions were kept 02-free as described in the text.
VANADIUM
INHIBITION
OF
tion and arsenate inhibition exhibit similar curves at 45°C with the pH of maximum binding to the enzyme being shifted to around pH 7.5 (8). Other ions of similar size and shape to phosphate (101-, MnO,-, C104-, CrOd2-) bind relatively weakly or at different sites than phosphate (9). Vanadium(V) (VO,-) appears to exhibit all the properties which would be expected for rather potent specific binding to alkaline phosphatase at the phosphate site. OxovanadiumUV) ion W02+) also proved to be a strong competitive inhibitor (Ki = 1.6 X 10e6 M, pH 8.0, 25”C, O-02 M Tris) of E. coli alkaline phosphatase (Fig. 5) and binds as tightly as phosphate. In barbital buffer, however, V02+ proved to be an even more potent inhibitor (Ki = 4 x lo-’ M, pH 8.0) and to bind very tightly over the pH range 7-9. Thus V02+ binds about four or five times better than VO,(and Pi> at pH 8 and one to two orders of magnitude better at pH 7.0. The pH-Ki profile (Fig. 3) differs from those of VO,and phosphate in exhibiting no apparent decrease in binding in the pH range 7-8.5. In the case of phosphate the decrease may be due to the protonation of the dianion with decreasing pH. A more general explanation might be protonation of an enzyme residue to which V02+ binding is insensitive. The binding of V02+ is apparently competitive in the pH range 7.0-9.5 in barbital buffer, as the lines on the inhibition plots (Fig. 6) intersect near 1.0. Theorell plots for V02+ exhibited a linear dependence on phosphate concentration at V02+ concentrations of about 5 PM or less with slopes nearly parallel (similar to Fig. 4 for VO,-). At higher V02+ concentrations, however, nonlinear plots were obtained. One possible explanation of these observations is that the V02+ may be binding much less tightly to other nonspecific sites on the enzyme, and this becomes evident only at higher V02+ levels. Other inhibition studies with V02+ have been done with ribonuclease (1) and acid phosphatase (5), both of which bind phosphate rather weakly. In these cases V02+ binds about three orders of magnitude better than phosphate and has been proposed to resemble the structure of the transition state (in ribonuclease complexed with uri-
ALKALINE
PHOSPHATASE
37
dine). No pH-Ki profiles on V02+ inhibition have been reported so it is not known whether the binding of this ion is sensitive to the ionization of enzyme groups in these two examples. In the case of E. coli alkaline phosphatase, phosphate is a very potent product inhibitor and one of the few good competitive inhibitors of this enzyme (6, 9). Oxovanadium binds one to two orders of magnitude better than phosphate, depending upon the pH, and an order of magnitude better than V02+ binding to wheat germ acid phosphatase. Both acid and alkaline phosphatases catalyze reactions proceeding via covalent phosphoryl-enzyme intermediates. A reasonable postulated transition state is a trigonal bipyramidal pentacovalent species with H,O entering and the alcohol (serine, alkaline phosphatase) or amine (histidine, acid phosphatase) leaving. Vanadate can form chelates with a variety of oxygen, nitrogen, and sulfur ligands (12) and usually adopts a distorted octahedral configuration or a trigonal bipyramidal one (10, 11). The bond angles and lengths of these complexes can vary, but for a typical V02+ complex they can be comparable to those of a pentacovalent phosphorous species (1). The potent competitive inhibition by V02+ suggests that this ion can form chelates at the enzyme active site which may bear some resemblance to the metastable intermediate occurring during the hydrolysis of the phosphate ester or phosphoryl enzyme. A similar proposal has been made for ribonuclease (1) and recently for acid phosphatase (5). REFERENCES 1. LINDQUIST, R. N., LYNN, J. L., AND LIENHARD, G. E. (1973) J. Amer. Chem. Sot. 95, 8762. 2. BENKOVIC, S. J., AND SCHRAY, K. J. (1973) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 8, p. 201, Academic Press, New York. 3. FITZGERALD, J. J., AND CHASTEEN, N. D. 11974) Biochemistry 13, 4338. 4. DEKOCH, R. J., WEST, D. J., CANNON, J. C., AND CHASTEEN, N. D. (1974) Biochemistry 13,4347. 5. VAN ETTEN, R. L., WAYMACK, P. P., AND REHKOP, D. M. (1975) J. Amer. Chem. Sot. 96, 6762. 6. REID, T. W., AND WILSON, I. B. (1971) in The
38
LOPEZ,
STEVENS
Enzymes, (Boyer, P. D., ed.), 3rd ed. Vol. 4, p. 373, Academic Press, New York. 7. WESTHEIMER, F. H. (1968) Accounts Chem. Res. 1, 70. 8. LAZDUNSHI, C., AND LAZDUNSKI, M. (1966) Biochim. Biophys. Actu 113, 551. 9. OHLSSON, J. T., AND WILSON, I. B. (1974) Biochim. Biophys. Actu 350, 48. 10. SELBIN, J. (1965) Chem. Rev. 65, 153. 11. CLARK, R. J. H. (1968) The Chemistry
of Titanium and Vanadium, p. 201, Elsevier, Amsterdam. 12. POPE, M. T., AND DALE, B. W. (1968) Quart. Rev. Chem. Sot. 22, 527. 13. NEU, H., AND HEPPEL,
L. (1965) J. Biol. Chem. 240, 3685. 14. TORRIANI, A. (1968) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.),
AND LINDQUIST Vol. 12B, p. 111, Academic Press, New York. 15. SIMPSON, R. T., AND VALLEE, B. L. (1968) Biochemistry
7, 4343.
16. SCOTT, W. W. (1962) in Standard Methods of Analytical Chemistry (Furman, N. H., ed.), 6th ed., Vol. 1, p. 1211, Van Nostrand, Princeton, N. J. 17. BRITTON, H. T. S. (1934) J. Chem. Sot., 1842. 18. DEAN, G. A., AND HERRINGSHAW, J. F. (1963) TuZuntu 10, 793. 19. PLOCKE,
D. J., LEVINTHAL,
C., AND VALLEE,
B.
L. (1962) Biochemistry 1, 373. 20. SNYDER, S. L., AND WILSON, I. B. (1972) Biochemistry 11, 1616. 21. NEUMANN, H., BOROSS, L., AND KATCHALSKI, E. (1967) Biochim. Biophys. Acta 113, 551. 22. THEORELL, H., AND YONETANI, T. (1964) Arch. Biochem. Biophys. 106, 243.
OF
BIOCHEMISTRY
Vanadium
AND
BIOPHYSICS
ion Inhibition V. LOPEZ, of Chemistry,
Department
175, 3138 (1976)
of Alkaline Phosphatase-Catalyzed Ester Hydrolysis1 T. STEVENS, San
Francisco
AND
State
Phosphate
R. N. LINDQUIST
University,
San Francisco,
California
94132
Received October 20, 1975 OxovanadiumW) ion (V02+) and vanadium(V) ion (VO,-) are potent competitive inhibitors of the Escherichiu co2i alkaline phosphatase-catalyzed hydrolysis ofp-nitrophenyl phosphate. The dissociation constants (KJ of the enzyme-inhibitor complexes have been determined as a function of pH over the pH range 7.0-10.0. Vanadium(V) & zz 2.2 x 10m6M, pH 8.0, Tris buffer) binds about as well as inorganic phosphate and has a pH-Ki profile similar to that of phosphate. OxovanadiumUV) ion binds about one to two orders of magnitude better than phosphate over this pH range (K, = 4 X, lo-’ M, pH 8.0, barbital buffer). Theorell-Yonetani plots show that vanadium(V) binds mutually exclusively with inorganic phosphate, and VO*+ also appears to bind in this manner at lower inhibitor concentrations. Tris buffer lowers the effectiveness of VO*+ as an inhibitor by a factor of four to five. These observations indicate that VOI- can bind quite specifically at the phosphate-binding site on the enzyme. The more potent inhibition by V02+ suggests that the ensyme-complexed inhibitor may have some resemblance to the metastable intermediate formed during the hydrolysis of phosphate esters.
Oxovanadium(IV) and vanadium(V) ions complexed with uridine are potent’inhibitors of ribonuclease and bind greater than three orders of magnitude more tightly than the corresponding substrate molecule uridine 2’,3’-phosphate (1). The ability of vanadium to form complexes of a trigonal bipyramidal nature led to the proposal that the vanadium complexes were acting as transition state analogs by virtue of their possible structural resemblance to the substrate portion of the transition state for ribonuclease-catalyzed hydrolysis of uridine 2’,3’-phosphate (1). Enzymatic phosphor-y1 transfer reactions in general are considered to proceed through a transition state involving a trigonal bipyramidal pentacovalent species in which entering and leaving groups occupy apical positions (2). If oxovanadium ions or their enzyme-chelated forms can be shown to be analogs of such metastable intermediates, then V02+ and VO,- ions have a wide potential in structural and mechanistic investigations of enzyme* This
work
was
catalyzed phosphate ester hydrolysis reactions, which are numerous in biological systems. Oxovanadium(IV) has excellent electron spin magnetic resonance properties, and Chasteen and co-workers (3, 4) have recently used V02+ to probe the active site of carboxypeptidase and carbonic anhydrase by replacing the zinc ion in the native enzymes. While our studies were in progress Van Etten et al. (5) reported that V02+ and other transition metal ions are excellent reversible competitive inhibitors of acid phosphatase. This enzyme hydrolyzes phosphate esters via a covalent phosphoryl-enzyme intermediate where an Nphosphorylhistidine (enzyme) is thought to be involved. Alkaline phosphatase (EC 3.1.3.1) is a metalloprotein with a broad substrate specificity, which catalyzes the hydrolysis
supported by NIH Grant No. GM
20817 to R. N. L. 31 Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
32
LOPEZ, STEVENS
AND LINDQUIST
phosphate, 25°C) monitored at 400 nm is about 25-30 of a number of phosphate esters presump,mol of substrate hydrolyzed per minute per milliably via a phosphoryl-enzyme intermediate where an active-site serine residue is gram of protein in agreement with Simpson and phosphorylated (6). The transition states Vallee (15). The final preparations were stored in for the formation and decomposition of this 0.01 M Tris at pH 8.0 at 4°C. Enzyme activity decreased only slightly over a period of 6 months. phosphoryl intermediate should resemble Disodium p-nitrophenyl phosphate was pura trigonal bipyramidal pentacovalent spe- chased from Sigma Chemical Company. Stock solucies in which the entering and leaving tions of oxovanadium(IV) were made by dissolving groups are axial (2, 7). There are few po- Fisher reagent grade VOSO,.2H,O in 0.01 M HCl. tent competitive inhibitors of alkaline The V(IV) concentration was determined by titraphosphatase aside from inorganic phos- tion of the stock solution with KMnO, (16) and was approximately 80% of the value expected on the phate and arsenate (6, 8). Recent studies by Ohlsson and Wilson (9) have shown basis of the weight and the empirical formula. Stock that periodate is the most potent reversi- solutions of V(V) were prepared by dissolving Fisher-purified NH,VO, in hot water; immediately ble competitive inhibitor of this enzyme upon cooling the pH was adjusted to 8.0 with NaOH. yet discovered (Ki = 0.5 x 10m7M, pH 8.6). All other chemicals were reagent grade and were This ion, however, binds at a different site obtained commercially. All solutions were prepared than phosphate and has a different pH-Ki from glass-distilled deionized water. Since V(IV) is profile than for phosphate or for substrate oxidized by molecular oxygen to V(V) at pH values binding. greater than about 4 (17, la), all solutions used in Oxovanadium(IV) ion (V02+) is known experiments with V(IV) were prepared from O,-free to form five-coordinate complexes with wa- water, and purified nitrogen or argon was passed ter and other ligands (10, 11). It is possible through them. The solutions were transferred using pipets and syringes that had been flushed with nithat V02+ could rapidly and reversibly form a chelated complex with the enzyme trogen. which resembles in some manner the tri- Kinetic Measurements gonal bipyramidal transition state in the The initial rates of hydrolysis of p-nitrophenyl alkaline phosphatase-catalyzed hydrolysis phosphate were measured spectrophotometrically of phosphate esters. It is also possible that using a thermostated (25°C) Gilford 240 spectrophovanadium(V) might adopt a trigonal bi- tometer with a Radiometer REC 51 recorder and a pyramidal structure since crystalline hy- 0.0-0.10 full scale absorbance mode. The reaction contained 2.8 ml of buffer and 200 pl comdrated metavanadates (VO,- * H,O) are solution prised of substrate, water, and inhibitor. The reacfive-coordinate with oxygen atoms, and tion was initiated by the addition of 10 ~1 of stock the geometry is approximately trigonal bi- enzyme solution to the cuvette and monitored at pyramidal (12). This paper describes the 400 nm for several minutes. Alternatively, in reacpotent inhibition of Escherichia coli alkations where inhibitors were present, the substrate line phosphatase by oxovanadium(IV) and was added last to initiate the reaction. Typically, an absorbance of 0.01 to 0.03 was needed to determine vanadium(V) ions. EXPERIMENTAL
PROCEDURES
Materials The enzyme was prepared from E. coli by the osmotic shock technique of Neu and Heppel (13) and purified by chromatography on DEAE-cellulose using a sodium chloride gradient (14). Alternatively, E. coli alkaline phosphatase chromatographically and electrophoretically purified was purchased from Worthington Biochemicals (BAPF). This preparation had comparable activity to that isolated and gave similar K, and KS values in the different buffers used. Enzymatic activity of such preparations (pH 8.0, 1.0 M Tris?, 1 x 1O-3 up-nitrophenyl 2 The abbreviations used are: PNPP, p-nitrophenyl phosphate; Tris, tris(hydroxymethyl)aminomethane; DEAE-, diethylaminoethyl.
the initial slopes. By monitoring the first few percent or less of the reaction, substrate hydrolysis and inorganic phosphate inhibition were kept to a minimum. The pH of the reaction mixture was measured at the end of each kinetic run. The initial velocity values were determined using the extinction coefficients determined for the p-nitrophenolate anion in the two buffers used. The reaction solutions were diluted 1:l with 0.1 N NaOH after completion and the A, values thus obtained (e400 = 1.84 x lo4 M-I cm-‘, 0.02 M Tris, 0.5 M KCl; l d0,,= 1.97 X 104 M-l cm-‘, 0.02 M barbital, 0.5 KCl). The presence of V02+ or VO,- in the solution did not alter these extinction coefficients, The Henderson-Hasselbalch equation was used to determine the percentage ofpnitrophenolate species present at each pH value assayed, and the observed change in absorbance was corrected accordingly. In addition, rates were nor-
VANADIUM
INHIBITION
OF ALKALINE
malized from day to day to a standard value of enzymatic activity which was checked at the beginning and the end of each set of kinetic measurements. The protein concentrations of the stock enzyme solutions were determined spectrophotometritally according to EjB. (278 nm) = 7.2 (19). Special precautions were taken to exclude oxygen in all assays involving VO*+. The cuvette was initially flushed with purified nitrogen, and the buffer, enzyme, and VO*+ solutions were added under a flow of nitrogen. After flushing the solution with nitrogen for a minute, the cuvette was stoppered and equilibrated in the thermostated spectrophotometer compartment for several minutes, and then substrate solution was quickly added to initiate the reaction. The stock solutions were all flushed with nitrogen afier every few runs and kept stoppered. No detectable enzyme inhibition was found with sulfate ion at concentrations up to 1O-3 M Na,SO,. Values of K, for p-nitrophenyl phosphate were determined at several pH values (25°C) in 0.02 M Tris-HCl and 0.02 M ethanolamine and 0.02 M barbital, all in 0.5 M KCl. In addition, the Ki for phosphate was determined at two different substrate concentrations (1 x 10m4and 5 x 10m5M) using six concentrations of P, (1 x 1O-5-7.5 x 10m5M) in 0.02 M TrisHCl, 0.5 M KC1 buffer, pH 8.0 and 25°C. The enzyme concentration was 0.43 pg/ml. At each substrate concentration the quotient of the uninhibited and inhibited rate (V,,/V) was plotted against the inhibitor concentration. These plots gave straight lines intersecting at 1.0 as expected for a competitive inhibitor. The Ki value determined from these plots (Ki = 1.5 x 1Om6Ml is in excellent agreement with a similar determination by Snyder and Wilson (K, = 1.3 2 0.1 x 10m6M) (20). All intercepts and slopes were determined using an unweighted least-squares program.
I I O7
PHOSPHATASE
I 9
I 9
IJ IO O
PH
FIG. 1. Enzyme activity as a function of pH in 0.02 M barbital, 0.5 M KC1 buffer (Ml and K, as a function of pH in 0.02 M Tris, 0.5 M KC1 (O), 0.02 M ethanolamine, 0.5 M KC1 (01, and 0.02 M barbital, 0.5 M KC1 (0). The r’action mixtures contained 1 x lo+ to 5 x 10m6M ofp-nitrophenyl phosphate in a total volume of 3.0 ml with an enzyme concentration of 0.57 or 0.31 pglml (barbital buffer). Specific activities were measured at a substrate concentration of 1 x 10m3 M. Initial rates were calculated from the initial slopes of AA/At at 400 nm, 25”C, using the calculated E values and appropriate product ionization factors (see Experimental). Values of Km were calculated from plots of l/initial velocity vs l/[Sl.
obtained at pH 9.0 in both Tris and ethanolamine btier systems. RESULTS A plot ofK, vs pH (Fig. 1) shows that K, values increase as the pH is increased Effect of pH on PNPP Hydrolysis from 7 to 10, consistent with other pubThe enzymatic activity and substrate and inhibitor binding ability of E. coli al- lished data (8, 21). Strong product inhibition is an inherent difficulty in this K, kaline phosphatase are sensitive functions so that only approximate of the buffer and ionic strength of the reac- determination values are obtained (20). Three buffer systion medium (6,9, 20), and buffers such as tems were used (0.02 M buffer in 0.5 M Tris can markedly affect V,,,. The specific activity of the E. coli alkaline phos- KCl) to cover the pH range 7-10. It appears that the K,,, values do not change phatase-catalyzed hydrolysis of p-nitrophenyl phosphate was measured as a fimc- appreciably as a function of buffer at the used. The K,,, values obtion of pH over the range 7.00-10.00 in 0.02 concentrations M barbital, 0.5 M KC1 at 25°C (Fig. 1). A tained are comparable to previously retypical activity profile results with this ported values (20). buffer and a similar profile was obtained with Tris and ethanolamine buffers in the Inhibition of PNPP Hydrolysis by VanapH range 7-10 which was consistent with dium(V) previously reported values under similar Figure 2 presents kinetic data for the conditions (20, 21). Identical activity was inhibition of E. coli alkaline phosphatase-
34
LOPEZ,
STEVENS
AND LINDQUIST
determined from the slopes of the lines. As with the K, determinations, it appears that the Ki values determined for VO,- do not change appreciably in going from Tris to barbital buffer. At pH 8.0, Ki = 2.2 x lo+ M in 0.02 M barbital, 0.5 M KC1 (vs I& I I I = 2.9 X 1o-6 M in 0.02 M Tris, 0.5 M KCl). The values of Ki forVO,- as a function of pH are plotted in Fig. 3. The Ki between pH 8 and 9 is from 2.5 to 3.5 x lop6 M, a value in the same range as inorganic phosphate under similar conditions (6,20). The inhibition is independent of the length of time (at least up to 30 min) that the inhibitor is preincubated with the enzyme before the addition of substrate. In Fig. 4 the Theorell-Yonetani plot (22) p0Jo3'11 IO-% of reciprocal velocity vs phosphate concenFIG. 2. Inhibition of the alkaline phosphatasecatalyzed hydrolysis of PNPP by VOI- at pH 8.0 and tration in the presence of a fixed concen25°C; PNPP concentrations: 1 x lo+ M (O), 5 x 10-S tration of VO,- and PNPP gives a series of lines over a wide range of VO,M (W, 1 x lo-* M (O), and 2 x lo+ M (0). The parallel reaction mixtures contained 0.02 M Tris, 0.5 M KCI, concentrations (3-100 PM). Parallel lines and enzyme at a concentration of 0.5’7 rig/ml in a to- in a plot of this type indicate that the two tal volume of 3.0 ml. The quotient of the uninhibited inhibitors, Pi and VOS-, bind in a muand inhibited initial rates (VJV) is plotted against tually exclusive fashion. If the lines were the vanadium(V) concentration. All rate values not parallel, it would indicate cooperative, have been normalized to one concentration of en- antagonistic, or independent binding dezyme, and the initial velocities were calculated from pending upon where the lines intersected. absorbance changes of approximately 0.02 to 0.03 At the concentrations of NH,VO, used, esafXer addition of substrate to the preincubated reacsentially all of the vanadium(V) is present tion solution. as the VO,- species and not as a trimer or catalyzed hydrolysis of PNPP by VO,- at higher polymeric species (1). The Ki value pH 8.0. At each PNPP concentration the for VO,- calculated from these lines is very close to that calculated from the V,,/V plots quotient of the uninhibited and inhibited (Fig. 2). rate (V,,/V> gives a straight line intersectAlthough the VdV vs VO,- plots (Fig. 2) ing the ordinate at 1.0 when plotted were linear over a wide range of VO,against the inhibitor concentration. The concentrations, above an inhibitor conceninhibition appears to be linearly competitration of about 1.5 x lop4 M there was a tive over a wide range of VO,- concentradeviation from linearity as the plots tions, and the kinetics can be described by curved downward from the original slopes. the equation This could be a result of polymer formation V by vanadium(V) to a trimer. It has been K, + [PNPP] o=calculated that, in 1.5 x 10m4 M NH,VO, v %a(1 + vo,-/K,) + [PNPP] ’ solutions, about 95% of the V(V) is in the which can be simplified to give (20) monomer state. At increased concentrations more trimer will form, thus decreasl/Ki = [l + (PNPPIK,)] X slope, ing the effectiveness of the inhibition per where Ki is the dissociation constant for mole of VOB- present. the enzyme-inhibitor complex. Inhibition by VO,- appears to be competitive at all Inhibition of PNPP Hydrolysis by Oxovanadium(IV) pH values between 7 and 10. Two to four Initial studies of the inhibition of alkadifferent substrate concentrations were used at each pH. The values of Ki were line phosphatase hydrolysis of PNPP by
:
VANADIUM
I 8
1n10-1 7’
INHIBITION
I
9
OF ALKALINE
I I
IO
PH
FIG. 3. Semilogarithmic plots of K{ vs pH for the inhibition of alkaline phosphatase-catalyzed hydrolysis of PNPP by vanadium(V) (VOJ (D) in 0.02 M Tris, 0.5 M KC1 (pH 7-S) or 0.02 M ethanolamine, 0.5 M KC1 (pH 9.5-10.0) and for the inhibition by oxovanadium(IV) (VO*+) (0) in 0.02 M barbital, 0.5 M KC1 (pH 7.0-9.5) or in 0.02 M ethanolamine, 0.5 M KC1 (pH 10.0). For VO,- inhibition, conditions were as described in the legend to Fig. 1 except that inhibitors were added to give the following concentrations: PNPP, 1 x 10m4-5 x 1O-6 M; VOs-, 1.7 x 10vs-6.7 x 1Om6M; enzyme, 0.57 pg/ml. Kinetic runs using four different substrate concentrations were done at pH 7, 8,9, and 10 and two substrate concentrations for the other pH values. The results at each pH were plotted as in Fig. 2. The& values which are the dissociation constants of the enzyme-inhibitor complex were obtained as described in the text, and an average value at each pH is plotted. For VO*+ inhibition the reaction concentrations were: PNPP, 2.5 X 10m5 M; VO*+,
1.2
x
1O-6-2.4
x
PHOSPHATASE
35
determined in the same manner as described for VOB-. The value determined at pH 8.0 from three kinetic runs was 1.6 x 10e6 M. Thus V02+ is a potent competitive inhibitor of alkaline phosphatase. It binds about twice as tightly as VOB- under the same conditions and has a Ki equivalent to that determined for phosphate (1.5 x lo-+ M). In order to determine if there were any buffer effects on the V02+ inhibition the Ki was determined at pH 8.0 in 0.02 M barbital, 0.5 M KCl. The points for V02+ at pH 8 in Fig. 3 demonstrate that V02+ is a markedly better inhibitor in the barbital buffer system (Ki = 4 x lo-’ M) than in the Tris buffer (1.6 x 1O-6 M). It has recently been reported (9) that Tris buffer has a protective effect toward the inactivation of alkaline phosphatase by permanganate ion. It is possible that this same phenomenon accounts for the reduced binding of V02+ in the presence of Tris, although such an effect is not apparent with VO,- inhibition.
1Om5 M; enzyme,
0.31 pg/ml. The K< for V02+ inhibition at pH 8.0 in 0.02 M Tris, 0.5 M KC1 (0) is an average value from three runs at different substrate concentrations. The V02+ studies and KS determinations were done as above for VO,- inhibition. 000
VO*+ were done in 0.02 M Tris, 0.5 M KC1 ‘buffer at pH 8.0. Kinetic measurements at several different substrate concentrations yielded straight lines intersecting near 1.0 on the VdV axis (Fig. 5), indicating competitive inhibition. A similar study at pH 10 in ethanolamine buffer exhibited a linear relationship also (Fig. 5). The KG was
plosRmTg.lO-%l
4. Theorell-Yonetani plot of initial velocity versus phosphate concentration at varying concentrations of VO,-: no VO,- CO),3 phi CO),5 PM (Ml, 33 PM (Cl), and 100 PM (A), and fixed PNPP concentration (5 x 1Om6Ml, pH 8.0, 25”C, 0.02 M Tris, 0.5 M KCl. Reaction conditions were as described in the legend to Fig. 1 with an enzyme concentration of FIG.
0.31 /&g/ml.
36
LOPEZ,
STEVENS
AND LINDQUIST
the enzyme in Tris or barbital buffer. The parallel lines in the Theorell-Yonetani plot (Fig. 4) indicate that VO,- and phosphate bind to the enzyme in a mutually exclusive fashion. Prolonged preincubation of VO,- with the enzyme does not yield any progressive increase in inhibition, indicating that no oxidation of the enzyme is taking place. Since VO,- exists almost exclusively as the monomeric species in solution at the concentrations used in the inhibition studies, the above results suggest that it may be acting as a phosphate analog in binding very specifically to the site(s) on the enzyme which normally ligands the phosphate anion. In addition, the pH-K, profile for VO,inhibition (Fig. 3) shows a maximum binding of this ion near pH 8.5 and a marked decrease in binding at pH 7 and 10. A similar bell-shaped pH-Ki plot is exhibited of alkaline phosphaFIG. 5. Inhibition of alkaline phosphatase by (8) for the inhibition VO*+ in 0.02 M Tris, 0.5 M KCl, pH 8.0, PNPP, 2 x tase by inorganic phosphate at 25°C with a 10e5 M (O), and 5 x 1O-5 M (W) and in 0.02 Methanolminimum at pH 8.0-8.5. Phosphate inhibi30
I-
1
1
amine, 0.5 M KCl, pH 10.0, PNPP, 5 x 10e5 M (0). Reaction conditions were as described in the legend to Fig. 2.
1
Another possible explanation is that Tris forms a complex with VOZ+ in the concentration range studied and that barbital either does not complex or forms a much weaker complex. Kinetic plots for the inhibition of E. coli alkaline phosphatase by V02+ in the pH range 7.0 to 9.5 in barbital buffer (Fig. 6) exhibit a linear dependence on V02+ in the concentration range used. The lines intersect the VdV axis near 1.0 suggesting a competitive type inhibition. The Ki values in this pH range were calculated as previously described and are plotted in Fig. 3. DISCUSSION
Vanadium(V) ion (VO,-) and oxovanadium(IV) ion (V02+) are both potent inhibitors of E. coli alkaline phosphatase. The former ion clearly exhibits competitive inhibition with the phosphate ion over the pH range 7.0-10.0 (Figs 1 and 2) and binds about as well as inorganic phosphate ion at pH 8.0 (Ki = 2.2 x lop6 M for VO,- vs Ki = 1.5 x 1O-6 M for phosphate). In contrast to V02+, the VO,- ion binds equally well to
FIG. 6. Inhibition of alkaline phosphatase by VOz+ at pH 8.0 (W, pH 7.0 (01, and pH 9.5 (0) (least-squares line for pH 9.5 has a steeper slope than line for pH 7.0). The reaction mixtures contained a PNPP concentration of 2.5 x 10m5M, 0.02 M barbital, 0.5 M KCl, and enzyme at a concentration of 0.91 pg/ml. All reactions were kept 02-free as described in the text.
VANADIUM
INHIBITION
OF
tion and arsenate inhibition exhibit similar curves at 45°C with the pH of maximum binding to the enzyme being shifted to around pH 7.5 (8). Other ions of similar size and shape to phosphate (101-, MnO,-, C104-, CrOd2-) bind relatively weakly or at different sites than phosphate (9). Vanadium(V) (VO,-) appears to exhibit all the properties which would be expected for rather potent specific binding to alkaline phosphatase at the phosphate site. OxovanadiumUV) ion W02+) also proved to be a strong competitive inhibitor (Ki = 1.6 X 10e6 M, pH 8.0, 25”C, O-02 M Tris) of E. coli alkaline phosphatase (Fig. 5) and binds as tightly as phosphate. In barbital buffer, however, V02+ proved to be an even more potent inhibitor (Ki = 4 x lo-’ M, pH 8.0) and to bind very tightly over the pH range 7-9. Thus V02+ binds about four or five times better than VO,(and Pi> at pH 8 and one to two orders of magnitude better at pH 7.0. The pH-Ki profile (Fig. 3) differs from those of VO,and phosphate in exhibiting no apparent decrease in binding in the pH range 7-8.5. In the case of phosphate the decrease may be due to the protonation of the dianion with decreasing pH. A more general explanation might be protonation of an enzyme residue to which V02+ binding is insensitive. The binding of V02+ is apparently competitive in the pH range 7.0-9.5 in barbital buffer, as the lines on the inhibition plots (Fig. 6) intersect near 1.0. Theorell plots for V02+ exhibited a linear dependence on phosphate concentration at V02+ concentrations of about 5 PM or less with slopes nearly parallel (similar to Fig. 4 for VO,-). At higher V02+ concentrations, however, nonlinear plots were obtained. One possible explanation of these observations is that the V02+ may be binding much less tightly to other nonspecific sites on the enzyme, and this becomes evident only at higher V02+ levels. Other inhibition studies with V02+ have been done with ribonuclease (1) and acid phosphatase (5), both of which bind phosphate rather weakly. In these cases V02+ binds about three orders of magnitude better than phosphate and has been proposed to resemble the structure of the transition state (in ribonuclease complexed with uri-
ALKALINE
PHOSPHATASE
37
dine). No pH-Ki profiles on V02+ inhibition have been reported so it is not known whether the binding of this ion is sensitive to the ionization of enzyme groups in these two examples. In the case of E. coli alkaline phosphatase, phosphate is a very potent product inhibitor and one of the few good competitive inhibitors of this enzyme (6, 9). Oxovanadium binds one to two orders of magnitude better than phosphate, depending upon the pH, and an order of magnitude better than V02+ binding to wheat germ acid phosphatase. Both acid and alkaline phosphatases catalyze reactions proceeding via covalent phosphoryl-enzyme intermediates. A reasonable postulated transition state is a trigonal bipyramidal pentacovalent species with H,O entering and the alcohol (serine, alkaline phosphatase) or amine (histidine, acid phosphatase) leaving. Vanadate can form chelates with a variety of oxygen, nitrogen, and sulfur ligands (12) and usually adopts a distorted octahedral configuration or a trigonal bipyramidal one (10, 11). The bond angles and lengths of these complexes can vary, but for a typical V02+ complex they can be comparable to those of a pentacovalent phosphorous species (1). The potent competitive inhibition by V02+ suggests that this ion can form chelates at the enzyme active site which may bear some resemblance to the metastable intermediate occurring during the hydrolysis of the phosphate ester or phosphoryl enzyme. A similar proposal has been made for ribonuclease (1) and recently for acid phosphatase (5). REFERENCES 1. LINDQUIST, R. N., LYNN, J. L., AND LIENHARD, G. E. (1973) J. Amer. Chem. Sot. 95, 8762. 2. BENKOVIC, S. J., AND SCHRAY, K. J. (1973) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 8, p. 201, Academic Press, New York. 3. FITZGERALD, J. J., AND CHASTEEN, N. D. 11974) Biochemistry 13, 4338. 4. DEKOCH, R. J., WEST, D. J., CANNON, J. C., AND CHASTEEN, N. D. (1974) Biochemistry 13,4347. 5. VAN ETTEN, R. L., WAYMACK, P. P., AND REHKOP, D. M. (1975) J. Amer. Chem. Sot. 96, 6762. 6. REID, T. W., AND WILSON, I. B. (1971) in The
38
LOPEZ,
STEVENS
Enzymes, (Boyer, P. D., ed.), 3rd ed. Vol. 4, p. 373, Academic Press, New York. 7. WESTHEIMER, F. H. (1968) Accounts Chem. Res. 1, 70. 8. LAZDUNSHI, C., AND LAZDUNSKI, M. (1966) Biochim. Biophys. Actu 113, 551. 9. OHLSSON, J. T., AND WILSON, I. B. (1974) Biochim. Biophys. Actu 350, 48. 10. SELBIN, J. (1965) Chem. Rev. 65, 153. 11. CLARK, R. J. H. (1968) The Chemistry
of Titanium and Vanadium, p. 201, Elsevier, Amsterdam. 12. POPE, M. T., AND DALE, B. W. (1968) Quart. Rev. Chem. Sot. 22, 527. 13. NEU, H., AND HEPPEL,
L. (1965) J. Biol. Chem. 240, 3685. 14. TORRIANI, A. (1968) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.),
AND LINDQUIST Vol. 12B, p. 111, Academic Press, New York. 15. SIMPSON, R. T., AND VALLEE, B. L. (1968) Biochemistry
7, 4343.
16. SCOTT, W. W. (1962) in Standard Methods of Analytical Chemistry (Furman, N. H., ed.), 6th ed., Vol. 1, p. 1211, Van Nostrand, Princeton, N. J. 17. BRITTON, H. T. S. (1934) J. Chem. Sot., 1842. 18. DEAN, G. A., AND HERRINGSHAW, J. F. (1963) TuZuntu 10, 793. 19. PLOCKE,
D. J., LEVINTHAL,
C., AND VALLEE,
B.
L. (1962) Biochemistry 1, 373. 20. SNYDER, S. L., AND WILSON, I. B. (1972) Biochemistry 11, 1616. 21. NEUMANN, H., BOROSS, L., AND KATCHALSKI, E. (1967) Biochim. Biophys. Acta 113, 551. 22. THEORELL, H., AND YONETANI, T. (1964) Arch. Biochem. Biophys. 106, 243.
