Phosphate Ester Hydrolysis by. Purple Acid Phosphatase from. Bovine Spleen*. (Received for publication, May 13, 1991). John B. Vincent$, Michael W. Crowder ...
T H E JOURNALOF BlOLOGlCAL CHEMISTRY Vol. 266, No. 27, Issue of September 25, pp. 17737-17740, 1991 6 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Communication Evidence for a PhosphorylEnzyme Intermediatein Phosphate EsterHydrolysis by Purple Acid Phosphatase from Bovine Spleen* (Received for publication, May 13, 1991) J o h n B. Vincent$, Michael W. C r o w d e r , a n d B r u c e A. Averill5 From the Department of Chemistry, University of Virginia, Charlottesuille, Virginia 22901
The possibility of the existence of a covalent enzymephosphoryl intermediate, E-POs, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase(PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22%of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be ratelimiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 3"Po3 group of [y-"P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has beendetermined over the range pH 38; a feature with a pK, of -6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a twostem pseudo Uni Bi mechanism that utilizes acovalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.
The purpleacid phosphatases (PAPS)'constitute a class of enzymes that catalyze the hydrolysis of certain phosphate esters, including arylphosphates and di- and triphosphonucleotides (1, 2). The most thoroughly studied of these are mammalian enzymes, which possess a dinuclear iron active site. PAPs exist in two forms: a purple, oxidized one containing adiferric center, which exhibitslittle or nocatalytic activity, and a reduced, pink one containing a mixed valent Fe(II1)-Fe(I1) center, which is enzymatically active. Little is known regarding the mechanism by which PAPs catalyze the hydrolysis of phosphate esters. Inasmuch as PAPs are the only acid phosphatases yet identified that possess a metal
cofactor, and iron remains quite uncommon in enzymes catalyzing non-redoxreactions,the possibility arisesthat a novel metal-centered mechanism may be employed. The involvement of a metaphosphate intermediate has been postulated previously (3); in contrast, product inhibition datasuggest a two-step pseudo Uni Bi hydrolytic reaction mechanism (4). The goal of the present work has been todetermine whether the PAP from bovine spleen functions via a phosphoryl enzyme intermediate,as required by thetwo-step reaction mechanism. MATERIALS ANDMETHODS
The PAP from bovine spleen was isolated according to published procedures (5). Phosphatase activity using para-nitrophenyl phosphate (p-NPP) as substratewas determined as previously described (G), using minor modifications as described below. Burst experiments with oxidized protein were performed in 100 mM Tris buffer, pH 8.1, using 50-pl aliquots of enzyme solution and 1 ml of buffer with the appropriate substrate concentration. The concentration of the hydrolysis product para-nitrophenol (p-NP) was determined using e = 16,250 a t 410 nm. The absorbance at 410 nm was corrected for the contributionfromtheaddedprotein.For reduced protein, 10-pl aliquots of protein were used, and the proteinwas adjusted to pH9.1 by dialysis against 100 mM CHES, pH 9.1, prior to use. The concentration of p-NP was determined using t = 18,300 at 410 nm. Transphosphorylation reaction mixtures contained10 mMp-NPP, 1.4 M alcohol, and 100 mM MES buffer, pH 6.0. Inorganic phosphate was quantitated as previously described (7). The pH stability of the phosphoryl enzyme intermediate was examined using previously reported procedures (8). Briefly, to reduced and oxidized PAP, [?-'"PI ATP (0.5 mCi) was added and allowed to incubate for 10 min. The resultant proteinwas then loaded onto and run on a 12.5% acrylamide native gel for 2.5 h as described elsewhere.' The gel was cut into two pieces and soaked in 50% methanol containing 0.1 N NaOH for 1 h to remove unreacted substrate. One gel was boiled in 50% trichloroacetic acid, while the other was heated in 1 M NaOH a t 60 "C for 30GO min. The gels were frozen and exposed on Kodak XAR-2 film for 1-2 h and developed using a Kodak X-Omat M-2 processor. After autoradiography, thegels were stained with Coomassie Blue to ascertain that the disappearance of '"P was not due to protein diffusion from the gel during washings. Phosphatase activity for pH dependence studies usingp-NPP as substratewas determined using 100 mM buffer solutions (NaOAc, pH 3-6; MES,pH 5-7; Tris,pH 6-8) containing 150 mM KC1 to minimize ionic strength differences from pH adjustment. The reaction was terminated 1 min after the addition of enzyme by the addition of 1 ml of 0.4 N NaOH/2.5 ml of assay; the amount of p-NP formed was determined using t = 18,300 a t 410 nm. K,,, and V values were determined from Lineweaver-Burk plots. The stability of the enzyme in the high pH region (pH 7-8) was tested by exposing the enzyme to these pHvalues followed by readjustment of the pH to 6.0 andmeasuringtheresultant enzyme activity. All experiments were performed in triplicate. Oxidized enzyme was generated as previously described (5). RESULTSAND
DISCUSSION
To date all acid phosphatases whose hydrolysis reactions * This research was supported by United States National Institutes have been investigated in detail have been found to proceed of Health Research GrantGM 32117. The costs of publication of this via a two-step mechanism as depicted in Equation 1 (7, 9article were defrayed in part by the payment of page charges. This 11).(The abbreviation"PO4"is used without anyimplication article must therefore be hereby marked "advertisement" in accord- to the protonation stateof the anion.) ance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by United States National Institutes of Health Postdoctoral Fellowship GM 13500. $ To whom correspondence should be addressed. ' The abbreviations used are: PAP, purple acid phosphatase; pNPP, para-nitrophenyl phosphate; p-NP, para-nitrophenol; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid MES, 2-(N-morpho1ino)ethanesulfonic acid.
E
kl
+ ROPO:, ZSE.ROPO3 " k-I
.1 ROH
+
E-PO:, A+ H,O
E
+ PO,
(1)
'J. L. Orlando, T. Zirino, and B. A. Averill, manuscript in preparation.
17737
E-PO, in PAP Phosphate Ester Hydrolysis
17738
In this reaction mechanism, the second product, phosphate, is predicted to be a competitive inhibitor of the hydrolase activity, while the alcohol, the first product, is expected be to a poor inhibitor. In the case of bovine spleen PAP, PO, has been shown to be a mixed type inhibitor, binding to a competitive site (the active site) and a second, noncompetitive site with K, = 5.4 mM (5); 5 mM p-NP did not inhibit the enzyme (12), in accordance with the two-step mechanism. Theseproductinhibitiondata, while suggestive, donot absolutely establishthatthe hydrolysis reaction of PAPS proceeds via the mechanismof Equation 1. Consequently, the establishment of the existence of a covalent phosphoryl enzyme intermediate(E-PO:,) is required. Towardthisend, transphosphorylation and burst experimentswere performed. If the two-step mechanism is operative,a steady-state quantity of the covalent phosphoryl enzyme intermediate should accumulateandbesusceptibleto being trapped. Athigh concentrations, alcohols should competeeffectively with H 2 0 forthephosphoryl enzyme intermediate,resultinginthe formation of the corresponding phosphoalcohol (transphosphorylation). Results with bovine spleen PAP using several alcohols are presented in TableI. Phosphate was transferred t o all alcohols examined. Moreover, the presence of the nucleophilic acceptors was found t o accelerate the rate ofp-NPP hydrolysis compared with only H 2 0 serving as nucleophile, consistent with results of transphosphorylationstudies of other acid phosphatases (7, 13). The results of Table I also confirm that phosphate ester hydrolysisproceeds by P-0 bond cleavage, in agreement with thework of Komkova et al. (14), who showed that hydrolysis of ADP and p-NPP catalyzed by the bovine spleen enzyme in I80H2 results in ''0 incorporation into thereleased phosphate group. Under conditions where k, is rate-determining in Equation 1 (k:,> [E],, then the formation of the phosphorylenzyme intermediate(E-PO,)duringtheburstshouldbe stoichiometric, resulting in the generation of 1mol of alcohol/ mol of enzyme active site. As acid phosphatases previously studied have been shown t o possess acid-labile, base-stable phosphoenzyme intermediates (7, 9), burst experiments with reduced PAP were performed over the pH range of 6.0-9.1. Non-stoichiometric bursts were observed below p H 8, but at 9.1, nearly stoichiometric amounts of alcohol were produced at a series of substrate concentrations (Table 11). A representativeburstexperiment is shownin Fig. 1A. A small dependence of the magnitudeof the burst on[SI, is observed, as predicted by Equation 2 (15) (where B = concentration of ROH produced in the burst).
TABLEI1 Burst experiments with reduced and oxidized P A P p-NPfPAP p-NP [p-NPP],, mM
nmol
nmolfnmol
1.00 2.50 5.00 10.0
0.48 0.49 0.87 0.87
0.58 0.59 1.06 1.06
0.63 1.25 2.50 5.00 10.0 100
0.78 0.84 0.61 0.89 1.31 1.01
0.83 0.88 0.64 0.93 1.38 1.07
Reduced P A P
Oxidized PAP"
~~
I'
0.828 nmol of PAP; pH 9.1, 100 mM CHES buffer. 0.947 nmol of PAP; pH 8.1, 100 mM Tris buffer.
I 5 ~ 0 . ' 0 0 E 0 0 20.b 25.W 3700
-0.01 3 I I 0.60
I
I,, I ,
I I . I I I I
-9
time(sec)
B)
-
0.25
1
0.15
-
0.05
-
0 .d-
> k3 and that k3 is rate-limiting. Thus, since kcat = (kz/kp + k3)ka,kcat= k3. Using Lineweaver-Burk plots to deduce Vmax and consequently kc,, (kc,, = Vmax/[Elo),kcat for the reduced enzyme at pH 9.1 was determined to be 0.33 s", giving an
~
~
E-P03in PAP Phosphate Ester Hydrolysis
17739
6.00 7 approximate value that sets an upper limit for k3. Oxidized PAP, which is reportedt o be "inactive" (12), might be expected to have anappreciably slower rate of phosphoryl enzyme dephosphorylation because of the increase inpositive i charge at or near the active site. As shown in Fig. lB, the 4.00 ' '*\ oxidized enzyme is indeed active and displays a nearly stoichiometric burst at pH 8.1 (Table 11). A plot of 1/& versus > 1/[S],,again gives a value of 1.0, indicating that k3 is ratedetermining at this lower p H for the oxidized enzyme. This difference in pH dependence clearly indicates that the activity 2.00 of the oxidized enzyme does not result from tracesof reduced enzyme. k 3for the oxidized enzyme at pH8.1 is approximately 8.5 s-1. As has been stressed previously (16, 17), most earlier burst experiments on acid phosphatases a t alkaline pH values were performed under conditionswhere [SI,
