pre-steady-state and steady-state kinetic analysis of the low

THE JOURNAL OF BIOLOGICAL CHEMISTRY (c, 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol 266. No. 3, Issue of January 25, pp. 1516-1525.1991 Printed in U.S.A.

Pre-steady-state and Steady-state Kinetic Analysis of the Low Molecular Weight Phosphotyrosyl Protein Phosphatase from Bovine Heart*

(Received for publication, June 28, 1990)

Zhong-Yin Zhang and Robert L. VanEttenS From the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

The complete time course of the hydrolysis of p- nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 "C, a transient pre-steady-state "burst" ofp-nitrophenol was formed with a rate constant of 48 s-'. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s". Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vma, was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed "0 exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 X s" a t pH 5.0,37 "C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Mi- chaelis binding constant K, = 6.0 mM, the apparent K , = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-*) and dephosphorylation (k3 = 36.5 s-I) were determined under steady-state conditions with p- nitrophenyl phosphate at pH 5.0 and 37 "C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium "0 exchange between phosphate and water was 20.2 kcal/ mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.

Low molecular mass (18-kDa) acid phosphatases (ortho- phosphoric monoester phosphohydrolase (acid optimum); EC 3.1.3.2) are active phosphotyrosyl protein phosphatases. This type of enzyme has been shown to hydrolyze readily substrates

*This work was supported by the United States Public Health Service Research Grant GM 27003 from the National Institute of General Medical Sciences. NMR instrumentation was supported by the National Institutes of Health Grant RR 01077 from the Division of Research Resources and by NSF/BBS-8714258. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed.

that include phosphotyrosyl (but not phosphoseryl) casein, IgG, red cell band 3, angiotensin, tyrosine kinase P40, and epidermal growth factor receptor (1, and references cited therein; 2). Along with other structurally distinct members of the phosphotyrosyl protein phosphatase family, these en- zymes may be expected to play a key role in cellular regulatory processes, for example by acting as part of a regulatory cycle counteracting the protein tyrosine kinases.

Despite their obvious significance, there is only a limited amount of mechanistic and kinetic information available on how the low molecular weight phosphotyrosyl phosphatases function. Free sulfhydryls are known to be essential for en- zymatic activity, raising the possibility that cysteine functions as a nucleophile or as part of a regulatory site (1-4). An active site peptide containing cysteine and arginine has been identified ( 5 ) . When a high molecular weight acid phosphatase was incubated with 32P-labeled p-nitrophenyl phosphate, a phosphoenzyme could be trapped, and the active site residue that was phosphorylated was identified as histidine (6, 7); but such experiments have not been done on the low molecular weight phosphotyrosyl protein phosphatase. However, related work showed that the low molecular weight enzyme from bovine liver catalyzes a stereospecific phospho group transfer from phenyl ( R ) - [1s0,'70,180]phosphate to (S)-propane- 1,2-diol, with overall retention of configuration a t phosphorus (8). This is consistent with a double displacement mechanism, provided that each attack on phosphorus proceeds by an in- line S N ~ ( P ) mechanism. The low molecular weight phosphotyrosyl phosphatase has a narrow substrate specificity and is able to hydrolyze efficiently only aryl phosphate monoesters (including tyrosine and related peptide and protein deriva- tives) and FMN (1). The fact that phenyl phosphates as well as phosphotyrosyl peptides and proteins can serve as substrates opens up a useful experimental approach since leaving group effects can be investigated. The phosphotyrosyl protein phosphatase from bovine heart also catalyzes a phosphate transfer from p-nitrophenyl phosphate to a variety of alcohol acceptor molecules, and this makes it possible to examine partitioning of potential phosphoenzyme intermediates. The overall rate of hydrolysis is increased greatly by the presence of a nucleophilic acceptor, whereas the level of inorganic phosphate that is produced remains relatively constant (1). These observations are consistent with the existence of a covalent phosphoenzyme intermediate in the hydrolytic pathway and with the breakdown of this intermediate being the rate-limiting step.

In the present study, we provide extensive evidence in support of this hypothesis. Evidence is provided for the existence of a covalent phosphoenzyme intermediate in the catalytic pathway, the order of substrate binding and product release, the rate-limiting step, and the energetics of the en-

1516

Kinetic Analysis of a Phosphotyrosyl Protein Phosphatase 1517

zyme-catalyzed reaction. The results are obtained through the use of a variety of steady-state kinetic techniques including partition experiments, examination of the selective influences of a nucleophilic acceptor on kc,, and K,, studies of the effects of leaving groups on V,,,, and a demonstration of an enzyme- catalyzed l80 medium exchange between phosphate and water. Significant results from an examination of the pre-steady- state kinetics are also described.

EXPERIMENTAL PROCEDURES

Materials-The low molecular weight phosphotyrosyl protein phosphatase was purified to homogeneity from bovine heart (1). Bovine hearts were obtained from a local slaughterhouse. Anhydrous ethylene glycol was from Aldrich. p-Nitrophenyl phosphate, phenyl phosphate, 0-phosphotyrosine, a-naphthyl phosphate and 0-naph- thy1 phosphate were from Sigma. All other substrates were synthesized by the procedure described here. The buffers were prepared using twice-deionized and distilled water. All the other reagents were analytical grade and were used without further purification.

Synthesis of Aryl Phosphate Monoesters-Aryl phosphate monoesters were prepared as follows (9). The substituted phenol (20 mmol) was dissolved in dry pyridine (20 ml, distilled over dry KOH), and this solution was added slowly with stirring to an ice bath-cooled solution of POCI:I (20 mmol) in 30 ml of dry pyridine. The system was protected from moisture. After the addition was complete, the reaction was allowed to stand for another 40 min before the mixture was poured onto 40 g of ice. The solution was adjusted to pH 9 using cyclohexylamine, and the resultant precipitate was filtered and re- crystallized twice from 90% ethanol. The yields of the twice-recrys- tallized cyclohexylammonium salts were generally above 60%. The purities of these preparations were confirmed by measurement of melting point, '"P NMR, and inorganic phosphate assays. A total of lour aryl phosphate monoesters (2-chlorophenyl, 4-ethylphenyl, 4- bromophenyl, and 3-nitrophenyl phosphate) were synthesized in this way.

Inorganic Phosphate- Water Medium "0 Exchange-The phosphatase-catalyzed medium ''0 exchange reaction between inorganic phosphate and water was conducted in 100 mM acetate, 1 mM EDTA, I = 0.15 M , pH 5.0, buffer containing 17% D20, a t several temperatures. The reaction mixture contained 0.416 mg of homogeneous, low molecular weight, bovine heart phosphotyrosyl protein phosphatase (specific activity, 78 units/mg) and 50-60 mM "0-labeled inorganic phosphate at 95.1% isotope enrichment (10). "P NMR was used to follow the exchange process (10) based on the observation that the "0 isotope caused an upfield shift in the "P NMR signal (11). Spectra were taken on a Varian XL-200 NMR spectrometer using the following parameters: acquisition time, 3.7 s; pulse width, 7.5 ps; spectra width, 50 Hz; and line broadening, 0.05 Hz, operating in the broad band decoupling mode.

Pre-steady-state Kinetics-The pre-steady-state kinetic behavior of the low molecular weight phosphotyrosyl protein phosphatase- catalyzed hydrolysis of p-nitrophenyl phosphate was studied using a Hi-Tech stopped-flow spectrophotometer (dead time, 2 ms) with an observation cell length of 1.6 cm and operating at 425 nm. The extinction coefficient for p-nitrophenol a t pH 7.0, 4.5 "C in 100 mM 3,3-dimethylglutaric acid buffer was 5,630 M" cm". The reaction was carried out a t 4 5 ° C in 100 mM 3,3-dimethylglutaric acid, 1 mM EDTA, pH 7.0, buffer. The substrate concentration was 20 mM. The enzyme (specific activity, 114 units/mg) concentrations were 5 and 16.5 PM in two separate runs. Briefly, 2 X concentrated substrate (40 mM) and 2 X concentrated enzyme (10 or 33 FM) were kept in the same pH 7.0 buffer in two separate syringes and equilibrated at 4.5 "C for at least 30 min. The reaction was started by triggering the fast mixing device while the release of p-nitrophenol was monitored at 425 nm. The data were collected on a IBM-PC computer interfaced to the Hi-Tech stopped-flow spectrophotometer. The specific rate constants were analyzed by fitting the experimental data directly to the theoretical kinetic equation through the use of a nonlinear least squares regression procedure.

Steady-state Kinetics-The steady-state kinetic measurements were performed in 100 mM acetate, 1 mM EDTA, I = 0.15 M, pH 5.0, buffer (assay buffer, unless otherwise specified). The reaction mixture (400 pl) containing substrate was first incubated in a 37 "C water hath for about 5 min, and then the reaction was started by the addition of a catalytic amount of enzyme. After specific time intervals (usually 3-5 min, when less than 2% of substrate hydrolyzed), the

reaction was terminated by the addition of 1 ml of 1 M NaOH, and the amount of phenol produced was measured using a UV-visible absorption spectrophotometer. The molar extinction coefficients were as follows: for p-nitrophenol, E4115 = 18,000 M" cm"; phenol, E287 = 2,560 M" cm"; tyrosine, E2Y:I = 2,411 M-' cm"; and 0-naphthol, E346 = 2,780 M-' cm" in alkaline solution. For inorganic phosphate determinations, reactions were terminated by the addition of 0.2 ml of 10% trichloroacetic acid followed by the addition of a 0.5-ml mixture (composed of 0.2 ml of 2% ammonium molybdate and 0.3 ml of 14% ascorbic acid in 50% trichloroacetic acid), and then 1 ml of 2% trisodium citrate plus 2% sodium arsenite in 2% acetic acid was added. The color was developed for 30 min, and the absorption at 700 nm was measured on a Beckman DU-68 spectrophotometer. The amount of inorganic phosphate produced was calculated from the standard curve using KH2P0, as a standard (12).

V,,, and K,,, Determinations-At least nine different substrate concentrations ranging from 0.1 to 10 K , were prepared, and the initial velocities of the enzyme-catalyzed hydrolysis were measured in assay buffer a t 37 "C (unless otherwise specified). The V,,, and K, values were obtained by fitting the data directly to the Michaelis- Menten equation using a Hewlett-Packard HP-85 computer and the program KINFIT (13).

Product Inhibition-The product inhibition study was performed a t 37 "C in assay buffer with p-nitrophenyl phosphate as a substrate at concentrations ranging from 0.1 to 10 K,. At least nine measurements were made of initial velocities a t each of three different inhibitor (p-nitrophenol or inorganic phosphate) concentrations. The inhibition constant K, was evaluated on an HP-85 computer using a direct curve-fitting program ENZYME (14).

Partition Experiments-The partition experiments were carried out at 37°C in assay buffer in the presence of 1 M ethylene glycol. Four different substrates, p-nitrophenyl phosphate, phenyl phosphate, tyrosine phosphate, and 8-naphthyl phosphate, were used in these experiments. The reaction was initiated by the addition of a catalytic amount of enzyme, and the reaction was terminated by the addition of 1 M NaOH or 10% trichloroacetic acid for phenol or phosphate determination as described above. The amount of phosphate transferred to ethylene glycol was calculated from the difference between the phenol and the inorganic phosphate produced.

The Determination of Ks, k,, ke and kA/k3"The method of Berezin and Kazanskaya (15) was employed to determine the true enzyme substrate dissociation constant Ks and various individual rate constants for other catalytic steps. In brief, the hydrolysis of p-nitrophenyl phosphate was conducted in assay buffer, a t 37"C, in the presence of various phosphate acceptor (ethylene glycol) concentrations ranging from 0 to 2 M. Under the steady-state conditions, the rate of the reaction was followed by measuring the production of inorganic phosphate. A plot of [E]o/uoc,~z, versus l/[S], gave straight lines, corresponding to various ethylene glycol concentrations, and the lines intersect at a common point in the upper left quadrant a t which the abscissa l/[S], = -1/KS. Similarly, the ratio of the rate constants for the phosphotransfer to the ethylene glycol and the rate constant for hydrolysis by water, kA/ku, could also be determined by plotting [ E ] O / V O ( ~ , , uersus [acceptor]. In this case, the lines corresponding to different substrate concentrations intersected in the left upper quadrant a t a point that corresponded to -k3/kA on the abscissa [acceptor]. Since kcat = k2k3/(k2 + k:j) and K, = Ksk, / (k2 + kTj), it was possible to calculate k2 and k, by solving these two equations simul- taneously.'

Phosphotransfer Reaction-The reaction mixture (4 ml) contained 10 mM p-nitrophenyl phosphate and 1 or 2 M glycerol in assay buffer a t 37 "C; the solution was made 1.1 X lo-' M in enzyme to start the reaction. Aliquots were withdrawn at intervals and used for p-nitrophenol and inorganic phosphate measurement. The amount of phosphate transferred was taken as the difference between the amount of p-nitrophenol and inorganic phosphate produced.

RESULTS

Product Inhibit ion Study-Product inhibition studies showed that at pH 5.0,37 "C, in the absence of p-nitrophenol,

Portions of this paper (including an appendix of a complete derivation of the equations used here, as well as Figs. 1-3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

1518 Kinetic Analysis of a Phosphotyrosyl Protein Phosphatase

inorganic phosphate acts as a pure competitive inhibitor for the enzyme-catalyzed hydrolysis of p-nitrophenyl phosphate, with an inhibition constant K; = 2.0 mM (Fig. 1A)' whereas in the absence of inorganic phosphate, p-nitrophenol acts as a classic noncompetitive inhibitor, with an inhibition constant K, = 80 mM (Fig. 1B).

Michaelis-Menten Parameters and the Temperature De- pendence of kCar-An analysis of Michaelis-Menten kinetics was conducted for a number of substrates including p-nitrophenyl phosphate, phenyl phosphate, tyrosine phosphate, CY-

naphthyl phosphate, P-naphthyl phosphate, 2-chlorophenyl phosphate, 3-chlorophenyl phosphate, 4-ethylphenyl phosphate, and 3-nitrophenyl phosphate, at pH 5.0 and 37 "C. The Vmax and K, values are summarized in Table I. From Table I, it is seen that with the exception of a-naphthyl phosphate and 2-chlorophenyl phosphate, the aryl phosphate esters ex- hibit an effectively constant V,,,,, value.

The temperature dependence of the phosphotyrosyl protein phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate was investigated at pH 5.0 and 7.0. Values of kc,, were determined at four different temperatures from 5 to 37 "C, and an Arrhenius plot was made to determine the energy of activation E,. At pH 5.0, E, was 13.6 kcal/mol whereas at pH 7.0 it was 14.1 kcal/mol (Fig. 2).

Partition Experiments-In the presence of 1 M ethylene glycol, at pH 5.0, 37 "C, a constant ratio of 1.32 was observed for the phosphate transferred to the ethylene glycol compared with the inorganic phosphate that was produced (Table 11). This ratio was found with four different substrates, namely p-nitrophenyl phosphate, phenyl phosphate, tyrosine phosphate, and P-naphthyl phosphate.

TABLE I V,,, and K,,, values for selected substrates at p H 5.0, 37 "C

Phosphate ester Leaving

group Vmax kcat K, DK,

4-Nitrophenyl 3-Nitrophenyl 2-Chlorophenyl 3-Chlorophenyl n-Naphthyl @-Naphthyl Phenyl Tyrosine 4-Ethvlahenvl

pMlmin s"

7.14 58.0 34.2 8.38 56.5 33.4 8.48 5.3 3.1 9.08 46.7 27.6 9.24 2.0 1.2 9.24 50.0 29.5 9.99 46.5 27.5

10.07 45.9 27.1 10.19 51.5 30.4

m A4

0.38 1.10

1.54

1.20 3.60

1.74

22

21

16.0

TABLE I1 Partition experiments

The partition experiment was performed in assay buffer, pH 5.0, in the presence of 1 M ethylene glycol, 37 "C. The reaction was quenched a t 3, 5, 6 , and 4 min for p-nitrophenyl phosphate, phenyl phosphate, 0-tyrosine phosphate, and P-naphthyl phosphate, respectively. Similar experiments were also performed under two other conditions. In one case, all the substrates were kept at 4 mM concentration, and reaction was terminated at 2.5 min; in the other, the p - nitrophenyl concentration was 1 mM; phenylphosphate, 1 mM; tyrosine phosphate, 2 mM; and p-naphthyl phosphate, 10 mM, and the reactions were terminated a t 3 min. They gave essentially the same ratio of transfer to hydrolysis.

Phosphate ester Phenol Phos- Phosphate Ratio Of

concentration produced transferred transfer to produced hydrolysis nmollmin nmoljmin nmollmin

p-Nitrophenyl (1 mM) 28.9 12.5 16.4 1.31 Phenyl (5 mM) 14.3 6.21 8.09 1.30 0-Tyrosine (10 mM) 2.53 1.11 1.42 1.28 0-Naphthyl (5 mM) 27.3 11.4 15.9 1.39

Phosphotransfer Reaction-At pH 5.0 and 37 "C in assay buffer containing 1 or 2 M glycerol, the low molecular weight phosphatase catalyzed not only the hydrolysis reaction but also a phosphotransfer reaction from 10 mM p-nitrophenyl phosphate to glycerol (Fig. 3). The partitioning from hydrolysis to transfer was increased substantially at the higher glycerol concentration (compare Fig. 3, A and B ) . The reaction was linear up to 15 min in the presence of 1.1 X lo-' M enzyme.

Inorganic Phosphate- Water Medium lRO Exchange-The low molecular weight phosphotyrosyl protein phosphatase was found to catalyze a medium phosphate-water '*O exchange reaction. Experiments were carried out at temperatures of 7, 25, and 35 "C at pH 5.0 in 100 mM acetate, 1 mM EDTA (I = 0.15 M) buffer containing 17% D20. Spectra taken of control samples to which no enzyme had been added confirmed that the spontaneous exchange process was negligible. (Even at an elevated temperature such as 100 "C, the spontaneous ''0 isotope exchange between phosphate and water is extremely slow, with a pseudo-first order rate constant of 7.5 x s" (16).) Fig. 4 shows an example of the time course of the enzyme-catalyzed phosphate oxygen exchange reaction. 31P NMR reveals clearly the change in the isotope distribution as a function of time. The kc,, for the enzyme-catalyzed exchange process was calculated according to Eargle et al. (17). At temperatures of 7, 25, and 35T , t he kcat values were 1.31 X lo-*, 1.23 x lo-", and 3.51 X s-', respectively. The energy of activation for the enzyme-catalyzed exchange reaction was 20.2 kcal/mol, calculated from the slope of an Arrhenius plot. A kcat value of 4.47 X s" for 37°C was calculated from the Arrhenius plot. For the kinetic model in Scheme 1, the partition coefficient PC is defined as PC = k2/(k-, + k2) and is the ratio

kl k ,

k - , k-2 E + P , e E . P i + E - P + H 2 0

SCHEME 1

of the rate at which bound phosphate loses H20 in the exchange step compared with the sum of the rates at which bound phosphate loses HzO plus the rate of release of inorganic phosphate to the medium. It was determined to be 0.03 according to PC = 1/3(4 - kpjKo,/kAE), where KP~RO, is the

A B _iA 0.10 0.05 0.00 PPM 010 0 05 000 PPM

C D

O.;O 0.05 0.60 PPM O b Ob5 0.00 PPM

FIG. 4. Time dependence of the low molecular weight phosphotyrosyl protein phosphatase-catalyzed l 8 0 exchange re-

35 "C. The '"P chemical shift of the PIRO4 species has been set to action between inorganic phosphate and water at pH 5.0,

zero whereas the four IGO isotopomers are located at 0.021, 0.042, 0.063 and 0.084 ppm further downfield. Spectrum A illustrates the isotopic composition of the sample before addition of enzyme; spectra B-D were taken 48, 128, and 531 h, respectively, after the addition of enzyme.


40 t 30

[SI In

FIG. 5. The enzyme-catalyzed hydrolysis of p-nitrophenyl phosphate in the presence of phosphate acceptor ethylene glycol at pH 5.0, 37 OC, in assay buffer. a-d, ethylene glycol concentrations were 0, 0.5, 1, and 2 M , respectively.

pseudo-first order rate constant for the disappearance of P1’04, and kA6 is the pseudo-first order rate constant for the disappearance of the average isotope enrichment

4

jP’8Ojl6oq-/

4 2 P180;16O4-, AE = JCo

/=o

0 s j 5 4) (18). The Determination of K , kp, k,, and kA/k3-Pre-steady-

state kinetic measurements are generally used to obtain the individual rate constants of enzyme-catalyzed reactions. Un- der favorable conditions, however, similar information can be obtained by steady-state kinetic measurements in the presence of certain effectors that selectively affect separate stages of an enzyme-catalyzed reaction. The product inhibition pat- tern, transphosphorylation, partitioning, and phosphate oxygen exchange results already obtained for this enzyme may be interpreted in terms of the kinetic model shown in Scheme 2. In order to obtain many of the individual kinetic constants shown in this scheme, we utilized an approach2

E + p N P P E.pNPP + E - P2 E kl k2 k3

k-1 k-, k-3

- P , + E + P ; + p N P kq

k-4

SCHEME 2

similar to that used in a study of a-chymotrypsin-catalyzed ester hydrolysis reactions, in which a nucleophilic reagent was added to the reaction mixture in order to accelerate the deacylation of the acyl-enzyme intermediate (15).

In the present case, the Michaelis-Menten kinetic parameters of the bovine heart phosphotyrosyl phosphatase-catalyzed decomposition of nitrophenyl phosphate were altered markedly upon the addition of ethylene glycol (Fig. 5). Con- sequently, we studied the steady-state kinetics of the low molecular weight phosphatase-catalyzed decomposition of p - nitrophenyl phosphate in the presence of the phosphate acceptor, ethylene glycol, at pH 5.0, 37 “C, in 100 mM acetate, 1 mM EDTA, I = 0.15 M, by measuring initial velocities of phosphate release (uO(p,)). The equilibrium enzyme-substrate

The abbreviations used are: pNPP, p-nitrophenyl phosphate; pNP, p-nitrophenol.

dissociation constant KS = k-Jk1 was determined directly from the experimental data. Upon plotting [E]o/uo(P, ) against l/[S],, the lines corresponding to the different ethylene glycol concentrations intersected at a point in the upper left quadrant at which the abscissa 1/[Sl0 = -1/& (Fig. 6). KS was found to be 6.0 mM at pH 5.0, 37°C. The ratio kA/k3, where kA is the rate constant for the dephosphorylation of the phosphoenzyme intermediate by the added phosphate acceptor ethylene glycol, and k3 is the corresponding rate constant for reaction of the phosphoenzyme with water, was obtained by plotting the data as [E]o/uocr,, uersus [A]. The lines formed by data obtained a t different substrate concentrations intersected in the upper left quadrant at a point corresponding to -k,/kA on the abscissa [ A ] (Fig. 7). Under these conditions, kA/k:3 was 1.33 M-’. (For a detailed analysis of the kinetic equations under these conditions, see the Miniprint.)

By applying the steady-state assumption to [E - P ] (the phosphoenzyme intermediate), it may be shown that K,,, = Ksk,/(k2 + ks), and kc,, = k2k3/(k2 + k3). Since k,,, under these conditions was determined to be 34 s-l (Table 111), k , and k, could be solved from the equations above to give k, = 540 s-l and k3 = 36.5 s-I. The ratio of the rate constants for the phosphorylation of the enzyme and the dephosphorylation of the phosphoenzyme intermediate was obtained from k,/k, =

Pre-steady-state Kinetics-The entire pre-steady-state and the steady-state kinetic course of the hydrolysis of p-nitro-

K.s/K, - 1 = 14.8.

7 t ,p I

- 1 0 1 2 3 4 5 6 7

I E - ~ x I/CSI M”

FIG. 6. Plot of [&“Jo/vo versus l/[S], for the determination of the true enzyme-substrate equilibrium dissociation constant KS at pH 5.0, 37 “C. At the intersection point, l/[S], = -1/KS. 0, no acceptor; V, [A] = 0.5 M; A, [ A ] = 1 M; and 0, [ A ] = 2 M .

c 6 0 5

\

n 4 w x 3

m t3 - 2

1

or ’ * I -2 -I 0 1 2 3

[ A I M

FIG. 7. Plot of [EIo/uo uersus [A] for the determination of the ratio of alcoholysis to hydrolysis kA/ka. At the intersection point, - k , / k , = [A] = -0.75 M so that ka/k:i = 1.33 M” ( k A is a second order rate constant). 0, [ s ] = 0.16 m M ; 0, [SI = 0.40 m M ; V, [SI = 1.2 m M ; A, [SI = 2.0 m M .


TABLE 111 kc,, and K,* values forp-nitrophenylphosphate

in the presence of ethylene glycol In assay buffer, pH = 5.0.

Ethylene glycol k a t K"1 k c a t l K m

M s -1 mM M" s" 0 34.2 0.38 9.0 x 10' 0.5 52.6 0.59 8.9 x 10' 1.0 73.7 0.82 9.0 X 10' 2.0 98.8 1 .OS 9.1 x loJ

0

N In

* a a 1 . . , , , , , , , J

OO IO 20 10 X TIME (SI

a N * a a

IO X TIME(S1 FIG. 8. The stopped-flow spectrophotometric trace of the

phosphotyrosyl protein phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate at pH 7.0 and 4.5 OC. The enzyme concentrations used in the upper and lower examples were 5 and 16 p ~ , respectively.

phenyl phosphate catalyzed by the low molecular weight phosphotyrosyl protein phosphatase was monitored successfully at pH 7.0 and 4.5 "C. Using a Hi-Tech stopped-flow spectrophotometer and monitoring at 425 nm, a pre-steady- state burst of p-nitrophenol was observed in 100 mM 3,3- dimethylglutarate, 1 mM EDTA, pH 7.0, buffer (Fig. 8). The formation of the burst occurred with a rate constant kS = 48 s" followed by a slow steady-state breakdown of the phosphoenzyme intermediate, with a rate constant k3 = 1.2 s-', calculated from a nonlinear regression procedure. The size of the burst showed a proportional dependence on the enzyme concentration when the experiments were conducted at two different enzyme concentrations, 5 and 16.5 NM, respectively (Fig. 8). The burst corresponded to 73% of the expected active site concentration in each case. This was a highly significant result for the following reason. The substrate concentration was about 6.7 x K, and k2/k3 = 40. Under these conditions, the maximal burst = [E],, (k2/(k2 + kn))'/(l + K,,,/[S])2 would in fact be expected to be only 72% of the enzyme active site concentration. Thus, the burst was effectively stoichiometric and is consistent with the presence of a single active site/ enzyme molecule.

DISCUSSION

Product Inhibition Study-The phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate can be categorized as a one substrate/two products (p-nitrophenol and phosphate) reaction, or a uni-bi system, since the other substrate (water) is effectively invariant in concentration. The present results obtained with homogeneous enzyme show that in the absence of p-nitrophenol, inorganic phosphate acts as a competitive inhibitor relative to the substrate whereas in the absence of phosphate, p-nitrophenol acts as a noncompetitive inhibitor relative to the substrate. These results establish that the kinetic scheme of the enzyme-catalyzed hydrolysis of phosphate monoesters is an ordered uni-bi system (19), in which one of the products (p-nitrophenol) is released first followed by the release of the second product, inorganic phosphate, consistent with Scheme 2.

V,,, and Temperature Dependence-The Michaelis param- eter V,,, was determined for nine potential substrates at pH 5.0 and 37 "C. Seven of the nine substrates, including p - nitrophenyl phosphate, 3-nitrophenyl phosphate, P-naphthyl phosphate, 4-bromophenyl phosphate, 4-ethylphenyl phosphate, and tyrosine phosphate, showed almost identical V,,, values, despite the fact that the pK, values of the leaving groups ranged from 7 to 10. These results suggest that a common phosphoenzyme intermediate is formed during these reactions and that the breakdown of this intermediate is rate limiting since the relative stability of the phenolic leaving group has little effect on Vm,, (or kcaL). The enzyme showed low activity only with two aryl substrates (a-naphthyl and 2- chlorophenyl phosphate) in which steric hindrance renders the phosphorus less accessible to an attacking group or to some other essential catalytic group. The results are thus consistent with the nature of the catalysis being nucleophilic.

Temperature dependence studies at both pH 5.0 and 7.0 revealed nearly identical energies of activation for the rate- limiting step (13.6 and 14.1 kcal/mol, respectively). Together with pH dependence studies for this (results not shown) and a related enzyme (20), which showed a constant kc,, over the pH range 4-7.0, these findings indicate that the rate-limiting step does not change in this pH range and that it is most likely a rate-limiting dephosphorylation step. The results from the pre-steady-state kinetics at pH 7.0 support this conclusion.

Phosphotransfer and Partition Experiments-The ability to catalyze phosphate transfer and partition reactions has long been regarded as strong evidence that a phosphatase-catalyzed hydrolysis involves a phosphoenzyme intermediate. The information obtained from these experiments can also help to identify the rate-limiting step, namely whether it is the phosphorylation of the enzyme or the dephosphorylation of a phosphoenzyme intermediate. Two types of partition experiments are carried out. In one, measurements are made of the rates of formation of the products for one particular substrate in the presence of various acceptors and concentrations. In the other, the hydrolysis of a series of substrates is carried out in the presence of a phosphate acceptor.

Consider the first of these in more detail (Scheme 3, where E - P is a phosphoenzyme intermediate and A is an acceptor molecule).

k, Ks

/-* E + Pi E + p N P P = E . p N P P + E - P

k,

- P - A + E SCHEME 3

If the rate-limiting step is the formation of a covalent


intermediate, and the acceptor reacts with the intermediate after the rate-limiting step, it does not increase the overall rate of the destruction of the ester. Consequently, the sum of the rates of the formation of the two products (from hydrolysis and phosphotransfer) will be the same and will be equal to the rate of formation of the intermediate. Thus, the addition of a phosphate acceptor would not change the rate of formation ofp-nitrophenol but would decrease the rate of formation of inorganic phosphate because the new pathway competes to lower the level of E - P. In other words, the presence of the acceptor only changes the product distribution but not the overall rate of destruction of the substrate.

If, however, the rate-limiting step is the hydrolysis of the intermediate, then the presence of the acceptor will increase the rate of the breakdown of the intermediate and hence increase the overall reaction rate (since small acceptor alco- hols are usually better nucleophiles than water). Then, the addition of a phosphate acceptor would increase the rate of formation of p-nitrophenol in response to the new pathway, the level of E - P would not fall, and the rate of formation of inorganic phosphate would remain unchanged. This is the case for the low molecular weight phosphotyrosyl protein phosphatase. We found that the rate of disappearance of substrate was increased dramatically in the presence of various alcoholic acceptors, although the level of inorganic phosphate that was produced remained unchanged compared with the level of phosphate produced in aqueous solution in the absence of acceptor (1). Furthermore, the increase in the overall rate of hydrolysis was proportional to the concentration of the acceptor present. This suggests that the breakdown of the phosphoenzyme intermediate is the rate-determining step since there will be a constant level of phosphoenzyme intermediate E - P under the saturating conditions used in these experiments whereas the presence of a nucleophile such as a low molecular weight alcohol, which is a better nucleophile than water, will increase the breakdown of E - P and thus the overall production of p-nitrophenol. Since alcohol concentrations were low (0.5 or 2 M), the rate of hydrolysis k3

= k’:%[H20] was nearly the same as that in the absence of acceptor, resulting in a constant level of phosphate produced in each case.

The second general type of partition experiment is to follow the hydrolysis of a series of different substrates in the presence of a phosphate acceptor, i.e. to determine the partitioning of the intermediate between competing acceptors. Regardless of which step is rate limiting, a common intermediate that is formed from several different substrates must give the same ratio of products at a given concentration of acceptors. In the presence of 1 M ethylene glycol, we studied the partitioning of the intermediate between competing acceptors (ethylene glycol and water) using four different substrates, p-nitrophenyl phosphate, @-naphthyl phosphate, phenyl phosphate, and tyrosine phosphate. These experiments were performed under three different sets of conditions (Table 11). In all cases, we found a constant ratio of 1.32 between the phosphate transferred to ethylene glycol and the phosphate produced for all four substrates.

The constant ratio of alcoholysis to hydrolysis strongly supports the conclusion that a common covalent intermediate exists. I t is otherwise difficult to see how this uniformity of relative rates for four different substrates could result, The possibility that the added acceptor directly attacks the Mi- chaelis complex was ruled out since for the disappearance of pNPP, kCat/K,,, = (kz[H20] + k , [A] ) /Ks was not a function of the acceptor concentration (Table 111) (21). Thus, the constant V,,, values for a series of substrates, the phosphotrans-

fer activity, the overall increase in the rate of hydrolysis in the presence of phosphate acceptor, and the constant product ratio in the partition experiments all suggest the existence of a covalent phosphoenzyme intermediate E - P and that the rate-limiting step in the hydrolytic pathway is the dephosphorylation of this intermediate.

Inorganic Phosphate-Solvent Water ‘‘0 Exchange-Solvent medium isotope exchange reactions in which the replacement of an atom or a group in a substrate is detected using a suitable isotopic label serve as excellent tools in mechanistic chemistry. In the case of the phosphatase-catalyzed hydrolysis of phosphate monoesters, a phosphate-water ”0 exchange affords an alternative way of studying the terminal steps in the overall catalytic scheme. We found that the low molecular weight phosphotyrosyl protein phosphatase does in fact catalyze an “0 exchange between water and ‘*O-labeled inorganic phosphate. This is also consistent with the existence of a covalent phosphoenzyme intermediate since the exchange reaction is easily accommodated by a minimum two-step reaction sequence (Scheme 1). In order for this exchange to occur, the covalent phosphoenzyme intermediate E - P is first formed with an accompanying loss of water (“0 labeled in this case). From the observed P1xOj1604-j distribution as a function of time during the enzyme-catalyzed “0 exchange (Fig. 4), combined with the determination of a partition coefficient that is effectively zero (so that k-l >> k2, i.e. the Michaelis-Menten assumption), we conclude that this enzyme-catalyzed exchange process is a total random sequential in which only one oxygen atom is exchanged per productive encounter of the phosphate with the enzyme. This results in a sequential cascade in which P‘”0, goes initially to P“OL80:,, followed by dissociation of P, from the enzyme (22). Unlike alkaline phosphatase, which has a phosphate K, at alkaline pH of 1 O “ j M, the low molecular weight phosphotyrosyl protein phosphatase has a phosphate Ki of 2 X M at pH 5.0. Thus, it binds inorganic phosphate 3 orders of magnitude more weakly than does alkaline phosphatase. Combined with all the information available from partition and ‘*O exchange experiments, it is unlikely that the dissociation of inorganic phosphate from the Michaelis complex E . P, is a rate-limiting step in the normal hydrolytic pathway.

The Determination of K.?, ka, kn, and kA/k:,“If the phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate follows the scheme given in Scheme 2 and if the decomposition of the phosphoenzyme intermediate is the rate-limiting step in the presence of competing acceptors for phosphate (Scheme 3), then we may derive the rate expressions for kc,, and K,, under steady-state conditions according to the procedures used for the chymotrypsin-catalyzed ester hydrolysis (23) assuming that the acceptor A does not bind strongly to the enzyme. For the formation of inorganic phosphate, kcat = k2ka’[H20]/kl + k:%’[H20] + kA [A], measured by the production of phosphate; for the formation of p-nitrophenol, kc,, = (kZkR*[H20] + M A [A])/(k? + kR’[H20] + k~ [A], measured by the production of p-nitrophenol; and k , = Ks(ke’[H20] + kA [A])/(k2 + k3’[H20] + k~ [A]. In general, then, the addition of external acceptor changes both the catalytic constant and the Michae- lis constant (Fig. 5 and Table 111). If one plots [E]O/o,),,>,, against l/[S], a t various acceptor concentrations, the lines should intersect at a point in the upper left quadrant a t which abscissa l/[S], = -1/& (see “Results”). The intersection at this point is consistent with the assumption that k:? is the rate-limiting step in the proposed schemes (Schemes 2 and 3).

There are three possible relationships involving KS and Km (21).


1. When k2 << k - l , the Michaelis-Menten mechanism holds,

2. When kz = k-l, Briggs-Haldane kinetics hold, Ks < K,,

3. When there are intermediates (covalently or noncova- lently bound) that occur after the E.S complex, kcat and K,,, will be combinations of various rate and equilibrium constants, and the relationship Ks 2 K,,, is found in all cases. We found that at pH 5.0,37 "C, the true thermodynamic dissociation constant for the enzyme p-nitrophenyl phosphate complex (Ks) was 6.0 mM whereas the apparent Michaelis constant K,,, was 0.38 mM, consistent with the existence of at least one intermediate after the E .pNPP complex. In addition, from the same set of experimental data, if one plots [E]o/uo,p,, against [ A ] , the lines corresponding to different substrate concentrations will intersect a t a point in the left upper quadrant on the abscissa - [ A ] = &/kA (see ''Results''). This supports the analysis based on the assumptions that there is a phosphoenzyme intermediate, that the breakdown of the intermediate is rate limiting, and that there is no direct interaction between the enzyme and the acceptor molecules. We found that kA/k3 = 1.33 M-'. Therefore, at 1 M ethylene glycol concentration, k A [ A ] / k s = 1.33. This is identical to the result obtained from the partition experiment, which showed that at pH 5.0, 37 "C, in the presence of 1 M ethylene glycol, the average ratio of phosphate transferred to the phosphate produced was 1.32 for four different substrates.

Pre-steady-state Kinetics-Pre-steady-state kinetic anal- yses and burst titration experiments have been used to detect intermediates on reaction pathways, to measure their rates of formation and decay, and to quantitate enzyme active site concentrations. Although there were several earlier attempts to perform burst titration experiments using high molecular weight acid phosphatases (24-26), all of those experiments were done using conventional spectrophotometers with man- ual mixing. More importantly, they were done at alkaline pH under conditions where [SI << K,,,. As we have explicitly pointed out earlier, the validity of those experiments is doubt- ful (27). The amplitude of the burst B is given by the equation

K.s = K,,, = k-l/kl .

K m = (k2 + k- , ) /k , = Ks -t kz/kl.

(AY + 8 B = [El,

where [Elo is the stoichiometric enzyme concentration, and [SI, i s the substrate concentration (28). If [SI0 << K,, then the expected burst becomes far less than stoichiometric and may become (as in the case of the earlier experiments) too small to expect to detect. Instead, mixing artifacts predomi- nate.

The main difficulty in experiments with phosphatases hav- ing pH optima in the dilute acid region is the lack of a good chromogenic leaving group at acidic pH values where the apparent K,,, value is low (since such phosphatases generally act on the phosphate monoanion). If one performs the experiments with nitrophenyl phosphate at alkaline pH, one gets great spectrophotometric sensitivity; but in order to observe a meaningful burst, one has to use very high concentrations of substrate (because K, is so high at alkaline pH). The absorbance due to the substrate (not to mention its solubility) may make such experiments technically impossible. We reached a compromise at pH 7.0. At this pH, the K,,, is 3.0 mM, making it possible to saturate the enzyme. We followed the release of p-nitrophenol a t 425 nm, since at that wave- length the difference in absorbance between p-nitrophenol and the substrate p-nitrophenyl phosphate is maximized. At

4.5"C, the reaction was within the detection limit of the stopped-flow instrument, and we could successfully observe burst kinetics for the acid phosphatase-catalyzed hydrolysis of a phosphate monoester.

Because the enzyme is used in relatively large amounts and the events involving changes on the enzyme are in effect observed directly, these types of experiments appear convincing. However, the results must be interpreted with caution because the "burst" may be due to effects other than the accumulation of intermediates. Some of the potential sources of artifacts are enzyme conformational changes upon substrate binding, a rate-limiting dissociation of the product, and severe product inhibition (21). Our product inhibition study showed that neither of the two products is a strong inhibitor. As discussed earlier, the dissociation of the product phosphate is not likely to be rate limiting nor is the dissociation of the product phenols because the values of V,,, are independent of the nature of the leaving group. Finally, if the substrate binding causes an enzyme conformational change, the change must be completed within the dead time of the stopped-flow instrument. We have determined the kc,,, Ks, kp, and k3 independently a t pH 7.0 and 5 "C by steady-state kinetics in the presence of various concentrations of ethylene glycol. At 5 "C, pH 7.0, kc,, was 1.25 s-', K,,, was 3.0 mM, and KS was 126 mM. Thus, kp and ks could be calculated according to equations kc,, = kzk3/(k2 + k3) and K , = Ksk3/(k, + k3), yielding kp = 52 s-' and k3 = 1.28 s-', These values are very similar to those obtained from the stopped-flow experiment (see "Results"). Since the present experiments were performed under conditions in which IS] >> K,, k3 << k2, and the burst was proportional to the enzyme concentration, the burst is convincing and is due to the fast stoichiometric release of p-nitrophenol. The existence of a covalent phosphoenzyme intermediate is implicated strongly, and it is predicted that its breakdown is rate limiting in the pH range under study here.

An Energetic Diagram of the Reaction-At pH 5.0, 37 "C, we have determined separately KS = 6.0 mM, kz = 540 s-I, k3 = 36.5 s-*, k3 = 4.47 X s" (from the "0 exchange experiment), and Ki = 2.0 mM for inorganic phosphate. The Bronsted selectivity coefficient P E ~ has been deduced from the literature data for the equilibrium constants for the hydrolysis of monophosphate dianions covering a wide range of oxygen donor acidity (BEQ = -1.35 -+ 0.06) (29). The correla- tion between log K' and the pK of the hydroxyl species XOH was linear.

= -1.35 k 0.06 pKXoH + 7.50 f 0.76

The activity of water is defined as unity. For p-nitrophenyl phosphate (pK = 7.14 for p-nitrophenol), K' = 7.26 x M*. At pH 5.0, we define Keg as the equilibrium constant for the hydrolysis of p-nitrophenyl phosphate.

From the Haldane relationship Keq = klk2k3k4/k-lk-zk-3k-4 = 1/Ks X k2/k-2 X k3& X K, (p , , . We obtained k-2 = 2024 M-' s-'. If we assume that both k , and k-4 are diffusion controlled (so that k , = k-4 = 4 X lo9 M" s-l), then k- , = 2.4 X lo7 s-' and kd = 8 x lo6 s-'. Using these results, an energetic diagram may be constructed for the enzyme-catalyzed hydrolysis of p- nitrophenyl phosphate at pH 5.0, 37°C (Fig. 9), assuming that the free energy of the system, free enzyme, and the free substrate are zero. The relative energy level for each species was calculated from the corresponding equilibrium constant


4

I6 - 14-

12-

10- - 8'

- -. 4 -

- E 6 -

8 2 - - cl -2-

-4 - -6 - -8 - -10-

-12-

Y E.P,

" 4

Reactlan Coordinate

FIG. 9. An energetic diagram for the low molecular weight phosphotyrosyl protein phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate at pH 5.0, 37 "C. The standard state is defined as the free enzyme and free substrate concentrations at 1 M. All the AGP and AGj values were calculated based on the experimentally measured equilibrium or rate constants, except for AGf and ACi which were estimated on the assumption that the enzyme binds nitrophenyl phosphate or inorganic phosphate at diffusion controlled rates.

Keq according to AGO = -RTlnk,, and the free energy of activation was calculated according to the transition state theory AGf = -RTln(kh/KT). For the kcst(hydrolysis) and kcat(exchange) steps, there is good agreement between the calculated AGf values and the experimentally determined energy of activation values E, (recognizing however that the two properties have the relation AGf = A H f - TAS#, A€€# = Ea): AG':, = 16.0 kcal/mol, E,, = 13.6 kcal/mol, AGf-3 = 21.5 kcal/ mol, and &(exchange) = 20.2 kcal/mol.

In conclusion, through the use of a variety of methods including pre-steady-state, steady-state, and "0 exchange studies, we have implicated the existence of a covalent phosphoenzyme intermediate on the pathway of the low molecular weight phosphotyrosyl protein phosphatase-catalyzed hydrolysis of phosphate monoesters, and we conclude that the breakdown of this intermediate is the rate-determining step. We also established that the enzyme-catalyzed hydrolysis reaction follows a kinetic scheme involving at least four steps: the fast association of the substrate to the free enzyme, followed by the attack of a nucleophilic active-site residue of the enzyme to form a covalent phosphoenzyme intermediate with the release of the phenolic leaving group, the phosphoenzyme intermediate E - P being then hydrolyzed slowly by water to give a Michaelis-Menten type complex E . Pi, and finally, the dissociation of inorganic phosphate from the enzyme. From these studies, we obtained the individual rate constants and equilibrium constants for the enzyme catalyzed reaction, and

constructed an energetic diagram for the system at pH 5.0 and 37 "C. Direct trapping experiments are in progress in an effort to identify the postulated nucleophilic residue.

Acknowledgment-We thank Professor Dale Margerum for the use of the stopped-flow spectrophotometer.

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Continued on next page.


I51 B Bo 7"-

?

pre-steady-state and steady-state kinetic analysis of the low

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