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guanine phosphoribosyltransferase from yeast by divalent zinc and nickel ions
Activation of Hypoxanthine/Guanine Phosphoribosyltransferase from Yeast by Divalent Zinc and Nickel Ions* Linda 2. Ali and Donald L. Sloan LZA, DLS. Department
of Chemistry and Ph.D. Program in Biochemistry, College of the City University of New York
The City
ABSTRACT We have observed previously that the reactions catalyzed by hypoxanthinelguanine phosphoribosyltransferase (HGPRTase) are activated by Mg(II), Mn(II), and Co(II), and we have defined the mechanism by which these activations proceed [Biochemistry 22, 3419-3424 (1983)]. A more extensive survey of the kinds of metal ions that will activate the HGPRTase catalysis now has been completed through the use of an HPLC assay procedure. Although Fe(I1) and Ca(I1) are unable to activate this reaction, a significant activation was achieved with the addition of spectroscopically pure Zn(I1) to the assay solution. In addition some IMP synthesis resulted from the addition of Ni(I1) to the assay mixture. Both the Zn(I1) and Ni(I1) kinetic effects on HGPRTase over a limited metal ion concentration range have been analyzed through the use of curve-fitting exercises. These results, in addition to the similar pH profiles for the activations by Mg(II), Mn(II), Co(II), and Zn(II), suggest that all of these metal ions activate the HGPRTase-catalyzed synthesis of IMP by way of the same mechanism [model II as defined by London and Steck, Biochemistry 8, 1767-1779 (1969)], during which two divalent ions bind to the HGPRTase active site per molecule of PRibPP.
ABBREVIATIONS PRibPP, 5phosphoribosyl a-1-pyrophosphate; HGPRTase, hypoxanthinelguanine phosphoribosyltransferase; OPRTase, orotate phosphoribosyltransferase; PPase, inorganic pyrophosphatase; HPLC, high pressure liquid chromatography.
* This work was supported by NIH grants AM-20183 and RR-08168 (MBRS), and by awards from the CUNY Research Foundation. Address reprint requests to Dr. Donald L. Sloan, Department of Chemistry and Ph.D. Program in Biochemistry, The City College of the City University of New York, 138th Street at Convent Avenue, New York, NY 10031. Journal of Inorganic Biochemistry 28,407-415 (1986) 0 1986 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
407 0162-0134/86/$3.50
408
L. Z. Ali and D. L. Sloan
INTRODUCTION Hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) catalyzes the formation of either inosine monophosphate (IMP) or guanosine monophosphate (GMP), respectively, from phosphoribosyl pyrophosphate (PRibPP) and hypoxanthine or guanine [Eqs. (1) and (2)]. The kinetic mechanism of this enzyme isolated from yeast has been determined to be Ordered Bi Bi [l] and, as illustrated by Eqs. (3) and (4), the mechanism through which the enzyme is activated by magnesium ion’ has been elucidated 121. hypoxanthine + PRibPP = IMP + PPi, (1) guanine + PRibPP PRibPP(A) E+M
* GMP + PPi,
* (2)
+ M ’- M-PRibPP(MA),
(3)
MEM ..+Kz “2 ME Km,lc-\ MEMA-tproducts. K.s,\‘ /’ KI MEA /
(4)
Interestingly, we have observed that Co(II) and Mn(II), as well as Mg(II), activate the HGPRTase-catalyzed reactions [2], and we reasoned that other divalent metal ions might also serve as essential activators. In this paper we report the results of our examination of the effects of addition of Zn(II), Ni(II), Ca(II), or Fe(I1) to the enzyme assay solution. These studies were facilitated by the use of a previously designed HPLC assay procedure that allows us to determine if the substrates or products are being removed from the assay solution via complexation with metal ions, in addition to defining the rate of product formation [2]. MATERIALS
AND METHODS
Materials Baker’s yeast (Budweiser Brand) was obtained from Valente Yeast Inc. (Flushing, were NY). PRibPP (sodium salt), hypoxanthine, IMP, GMP, and triethanolamine supplied by Sigma Chemical (St. Louis). Chelex-100 ion exchange resin was purchased from Bio Rad Laboratories, whereas nickel chloride was supplied by Alfa Division, Ventrom Corp. (Danvers, MA). All other chemicals, including the chloride salts of zinc, iron, copper, manganese, magnesium, calcium, and cobalt were analytical grade. Methanol (Mallencroft reagent grade) was distilled prior to use during HPLC, and a Gelman “Water I” purifier was employed to prepare HPLCgrade water. Enzyme
Purification HGPRTase was isolated from yeast to electrophoretic previously published procedures [ 1, 31.
homogeneity
through the use of
’ The nomenclature for association and dissociation constants that was defined by London and Steck [Biochemistry 8, 1767-1779 (1969)] has been adapted for use in this report.
For example,
KO is an
association constant for the PRibPP-metal ion interaction [Eq. (3)] and is equal to [M-PRibPP]/ [PRibPP][M]. The parameter I&, is the dissociation constant for this interaction and is equal to the reciprocal of KO. Other binding events, defined by association constants K,, K,,, , KS, K,, K2 are illustrated in Eq. (4).
PHOSPHORIBOSYLTRANSFERASE
High-Performance
Liquid
ACTIVATION
409
Chromatography
A Waters Analytical HPLC Instrument equipped with a Model M45 solvent delivery system, model U6K sample injector, model 440 absorbance detector, and a Houston Omniscribe Recorder was employed in the enzyme assay procedure. A single 4 mm x 30 cm Waters uBondapak C1s column (equilibrated with 10% methanol) was placed on-line with the solvent delivery system at a flow rate of 1 ml/min. Samples (10 pL), from solutions containing the enzyme, substrates, and the appropriate concentration of one of the divalent metal ions, were injected into the column with a Hamilton 801 microliter syringe and eluted under 700-1OOO psi. Hypoxanthine and IMP in the eluent were detected at 254 nm with a 0.1 recorder setting. All of the solvents used in the chromatographic procedure were eluted by vacuum filtration through a 0.45-pm HA Millipore filter. Enzyme
Assay Procedure Measurements of the HGPRTase catalyzed reactions, and the activation of these reactions by increasing concentrations of divalent metal ions, were accomplished by using a modification of the method described by Flaks [3]. The assay solution consists of 0.2 ml hypoxanthine (54 PM final concentration), 0.4 ml of a PRibPP solution (100 PM final concentration), 0.1 ml of one of the standard divalent metal ion solutions, 0.1 ml of 20 mM triethanolamine (pH 7.8), and water to a final volume of 2.4 ml. The mixture was placed in a 38’ C water bath and the reaction was initiated by the addition of 1.6 ml of a dilute enzyme solution (approximately 1 milliunit). 2 Aliquots of this solution were removed at appropriate time intervals, and the reaction occurring in the samples was terminated by heating in a boiling water bath for 2 min. The samples were then clarified, first by centrifugation and then by passage through a 0.45-pm HA Millipore filter prior to the HPLC injection. The HPLC elution profiles (Fig. 1) were employed to determine the time course of the reaction as described previously [4, 51. During the kinetic analysis various concentrations of PRibPP and pH values between 6.0 and 9.5 were employed. These concentrations and pH values are listed in the text and figure legends. To ensure that no metal ion contaminents were present in the assay solutions prior to the addition of the appropriate divalent metal ion, all of the substrate and enzyme solutions, as well as the triethanolamine buffer, were eluted through Chelex- 100 minicolumns.
RESULTS
AND DISCUSSION
Survey of Activation
by Various
Metal Ions
The ability of Co(I1) to activate the synthesis of purine nucleotides [2] but not the OPRTase-catalyzed synthesis of orotidine monophosphate [7] prompted us to ask if other metal ions could serve as the essential activator of the synthesis of IMP. The results of a survey of some physiologically important metal ions are listed in Table I. As shown in Table 1, solutions of Ca(II) and Fe(I1) do not serve as activators of the HGPRTase-catalyzed reaction whereas some activation is provided by the addition of Ni(I1) to the assay solution. Interestingly, Zn(I1) possesses the ability to activate the enzymatic synthesis of IMP to the same extent as Mn(I1) and Co(I1). To our knowledge, this marks the first time that Zn(I1) has been observed to activate a 2 One unit of HGPRTase activity is defined as 1 pmole IMP formed/min.
410
L. Z. Ali and D. L. Sloan
I
IMP
I
I
II /j II
64
I hypoxanthine
I
20
Elution
Time
(min.)
FIGURE 1. HPLC assay procedure for the HGPRTase catalyzed synthesis of IMP. The initial reactant concentrations for this analysis were 54 pm hypoxanthine, 167 pm PRibPP, and 80 pm Zn(I1). The reaction conditions and HPLC elution conditions were described in the Methods
section. phosphoribosyltransferase. HGPRTase is reminiscent [S], which requires three examine the activation of Activation
by Divalent
This less specific requirement for metal ions exhibited by of the activator requirements of inorganic pyrophosphatase metal ions at the active site. Therefore, we elected to HGPRTase by Ni(II) and Zn(II) in more detail.
Nickel Ion Solutions
The addition of spectroscopically pure Ni(II) to an assay solution containing reactants except a metal ion activator leads to a relatively slow but significant TABLE 1. Observed
Optimal Velocity (V) and Optimal Metal Ion Concentration ([Ml) for Activation of the Hypoxanthine/Guanine Phosphoribosyltransferase Catalyzed Synthesis of IMP Metal ion
V
CallI) Co(H)
0.7
100”
0.4 0.7 0. I 0.7
700-I 100 100-600 400-1000 X0”
w
[Ml
Fc(II) Mg(II) Mn(I1) Ni(I1) Zn(I1)
a At metal ion concentrations reactant precipitation occurred
higher than the value shown, in the assay solution.
all of the synthesis
PHOSPHORIBOSYLTRANSFERASE
I 4-
I
I
ACTIVATION
411
I
A
A-
/_
1
10
lo2
lo3
NM Ni (II)
I 1
1
2
[MM Mg(ll)PRibPP]-’
FIGURE 2. (A) Initial velocity (u) measurements of Ni(II) activation (-•-) and Mg(II) activation (a) of the HGPRTasecatalyzed formation of IMP using the HPLC assay procedure. The concentrations of hypoxanthine and PRibP were 50 and 167 pm, respectively. (B) Double reciprocal plots of v I vs. the calculated reciprocal concentration of Mg(II)-PRibPP in the presence (-0-) and absence (-•-) of 200 pm Ni(I1). The reaction conditions and HPLC elution conditions were described in the Methods section.
of IMP (Fig. 2). The addition of concentrations of Ni(I1) higher than 0.8 mM resulted in a decrease in soluble hypoxanthine, as determined by a decrease in the HPLC elution peak amplitude of the filtered assay solution. In order to test whether this activation by Ni(I1) proceeds through a mechanism similar to the activation by Mg(I1) [2], a competition assay procedure was devised. As shown in Figure 2, a plot of the rate of IMP formation versus the calculated concentration (Z&, = 260 PM) of Mg(II)PRibPP [2], in the presence and absence of 0.2 mM Ni(II), defines a competition between the metal ions. This result suggests that the activation by Ni(I1) of the HGPRTase-catalyzed reaction proceeds through the mechanism shown in equation (4) and is defined by the kinetic equation below [Eq. (5)]. Theoretical curve-fitting exercises, making use of equation (4), were then employed to evaluate the appropriate affinity constants for the Ni(I1) activation (Table 2). The mechanism described by Eqs. (3) and (4) defines three general regions for a plot of Vversus [Ml: (1) an activation region where V increases with [Ml, (2) a region where V is independent of [Ml, and (3) an inhibition region where I/ decreases with increasing [Ml. For the activation by Ni(II), the only region that was observed was the activation region, and therefore KS and K,,, could not be evaluated.
v/ v,,, =
KcK, [Mlf [M-PRibPP] K, [Mlf (KS[PRibPP] + Km [M]f+ Kc [M-PRibPP]
+ 1) + 1
(5)
412
L. Z. Ali and D. L. Sloan
TABLE 2. Calculated
Constants for the Metal Ion Activation of the HypoxanthineBuanine Phosphoribosyltransferase Catalyzed Synthesis of IMP.”
Mn(II) Mg(II) Co(I1) Zn(I1) Ni(I1)
17 260 17 150’ 20’
22 19 22 -_d _d
1,100 900 1,100 -._d _d
6 21 5 20 20
8 11 8 50 10
0.1 3.2 0.1 15 0.4
6.2 1.50 6.2 290 10
y The definitions of these micromolar dissociation constants have been defined in a footnote to the text and by London and Steck [lo]. b The calculated parameters for Mn(II), Mg(II), and Ca(I1) and the methods for the complete error analysis of these interactions have been described previously by Ali and Sloan [2]. c These values of & were estimated from competitive effects of these metal ions on the paramagnetic effect of Mn(II) on the water proton longitudinal relaxation rates of PRibPP solutions. as defined by Victor. LeoMensah, and Sloan [7]. d Only the inhibitory region of the initial velocity-versus-[M(II)] curve can be used to define the value of these parameters, and since this region was not characterized for these metal ions, the values of I?, and x,,, used in the curve-fitting exercises were those determined for Mg(I1).
Activation
by Divalent
Zinc Ion Solutions
Activation of the enzyme-catalyzed formation of IMP by Zn(I1) was examined at three different PRibPP concentrations and a range of Zn(I1) concentrations. The rate decreases that were observed at concentrations beyond 100 ,wM were attributed to a loss of soluble base in the assay mixtures prior to the initiation of the reaction, since these decreases also occurred in assay solutions that contained no PRibPP. However, the concentration of hypoxanthine remained unchanged when the range of S-100 PM Zn(I1) was employed in the assay procedure, and theoretical curves that employed equation (5) were generated to describe these results (Fig. 3). The constants that resulted from these calculations have been compiled in Tables 1 and 2. These results reveal that Zn(I1) is an effective activator of the HGPRTase-catalyzed reaction, over this limited metal ion concentration range.
I
I
I / I I , /I
I
/
, , ( , I ,(
6-
” x10
1
10
MM Znlll)
lo2
FIGURE
3. Initial velocity (u) measurements of the Zn(I1) activation of the HPIGPRTase-catalyzed formation of IMP using the HPLC assay procedure. The concentration of hypoxanthine in the incubation solutions was 54 pm. The concentrations of PRibPP employed were 33 pm (-•-), 67 pm (-0-). and 167 /*rn (-0-). The reaction conditions and HPLC elution conditions were described in the Methods section.
PHOSPHORIBOSYLTRANSFERASE
ACTIVATION
413
pH Analysis The final question we wished to answer concerned the pH effect on the various metal ion activations. As shown in Figure 4, the pH where each of the activations is maximal is within the same range (pH 8) whereas at pH 9, Co(I1) and Zn(II), but not Mn(I1) or Mg(II), have lost the ability to activate this reaction. [In contrast to this enzymatic reaction, the optimum pH at which Zn(II) activates inorganic pyrophosphatase, pH 6, is much lower than the optimum pH values observed for Mn(II), Mg(II), and Co(II).] These results suggest that the mechanism by which each of these metal ions activates the enzymatic synthesis of IMP is the same. Moreover, the results do not reflect coordination of the metal ions with hypoxanthine, since the crossover coordination of Ni(I1) and Zn(I1) with the N-7 or N-9 positions of this base occurs at pH values of 7.1 and 6.7, respectively [9], and this coordination would inhibit nucleotide formation., Mechanism
by Which
HGPRTase
is Activated
by Metal Ions
The activation by a variety of divalent metal ions has been examined for two other yeast enzymes, orotate phosphoribosyltransferase (OPRTase) and inorganic pyrophosphatase (PPase) [7, 81. Although the reactions catalyzed by PPase and HGPRTase are quite different, the mechanisms by which they are activated are apparently similar, since the obligatory active form of PPase is also an M2E complex. Not surprisingly, both enzymes proceed with a broad metal ion specificity. In contrast, the OPRTasecatalyzed reaction, which is analogous to those catalyzed by HGPRTase, proceeds
FIGURE 4. The pH profiles for the activations of the HGPRTase-catalyzed synthesis of IMP by Mn(I1) (-0-), Zn(I1) (-A-), Co(U) (-A-), and Mg(II) (-•-) in the presence of 170 PM PRibPP. The concentration of each metal ion was maintained at 83 PM. The reaction conditions as well as the HPLC elution conditions were as described in the Methods section.
V
x10
I
I
I
1
7
8
9
PH
J
414 L. Z. Ali and D. L. Sloan
through the use of a Ping Pong Bi Bi kinetic mechanism and is activated by metal ions via a mechanism that provides a role for two metal ions but requires only one essential metal ion [7]. This activation is defined as Model III by London and Steck [lo], in which both the apo-enzyme (E) and holo-enzyme (ME) forms of OPRTase are active [Eq. (6)]. The common factor in the mechanisms by which HGPRTase and OPRTase are activated [Eqs. (4) and (6), respectively] is the requirement for a metal ionPRibPP complex (MA) as the true substrate [2. 71. ME = MEMA &t ES EMA
-+ products -+ products
(6)
If indeed the metal ions coordinate only with the enzymes and with the phosphate groups of PRibPP, then several mechanisms by which HGPRTase may utilize two metal ions to catalyze the formation of a nucleotide can be envisioned. During one such mechanism, the enzyme-bound metal ion would coordinate the 5 ‘-phosphate group and position it away from the ribose ring so that on-line displacements at the 1 ‘position would be facilitated. During an alternate mechanism, the two metal ions are positioned on opposite sides of the pyrophosphate group and not only neutralize in part the negative charges present on the pyrophosphate portion of PRibPP, but form an ionic bond with the negative charge that would result from an on-line pyrophosphate displacement by hypoxanthine. Both of these mechanisms would be consistent with the observation that high Mg(I1) and Mn(I1) concentrations, where ME. MzE, and MzPRibPP complexes would exist, are inhibitory [2]. The order in which transition metal ions coordinate ligands. the Irving-Williams order of stability constants [ 11, 12, 131, is Ni(I1) > Zn(I1) > Co(H) > Mn(II), whereas the order of affinity of metal ions for HGPRTase-bound PRibPP (Table 2) is estimated to be Co(H) = Mn(I1) > Zn(I1) = Ni(I1). This unusual order indicates that the coordination of these metal ions at the HGPRTase active site is to both amino acid residues and to the phosphate and pyrophosphate groups of PRibPP. Chemical modification studies of the essential amino acids of HGPRTase underway currently may reveal which residues coordinate these essential metal ions.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
L. Z. Ali and D. L. Sloan, J. Biol. Chem. 257, 1149-l 155 (1982). L. Z. Ali and D. L. Sloan, Biochemistry 22, 3419-3424 (1983). R. Schmidt, H. Weigand, and U. Reichert, Eur. J. Biochem. 77, 77-85 (1979). J. G. Flaks, Methods Enzymoi. 6, 144-148 (1963). D. L. Sloan, Adv. Chromatog. 23, 97-125 (1984). D. L. Sloan, L. Z. Ali, D. Aybar-Bat&a, C. Yan, and S. L. Hess. J. Chromatog. 316, 43-50 (1984). J. Victor, A. Leo-Mensah, and D. L. Sloan, Biochemistry 18, 3597-3604 (1979). K. M. Welsh, A. Jacobyansky, B. Springs. and B. S. Cooperman, Biochemistry 22, 2243-2248 (1983). R. B. Martin, Act. Chem. Res. 18, 32-38 (1985). W. P. London and T. L. Steck, Biochemistry 8, 1767-1779 (1969). H. Irving and R. J. P. Williams, J. Chem. Sot., 3192 (1953).
PHOSPHORIBOSYLTRANSFERASE
ACTIVATION
415
12. M. N. Hughes, Inorganic Chemistry of Biological Processes, Wiley, London, 1972, Chap. 3, p. 71. 13. L. G. Sillen and A. E. Martell, Stability Constants of Metal-Ion Complexes, The Chemical Society, London, 1964.
Received June 30, 1986; accepted July 9, 1986
of Chemistry and Ph.D. Program in Biochemistry, College of the City University of New York
The City
ABSTRACT We have observed previously that the reactions catalyzed by hypoxanthinelguanine phosphoribosyltransferase (HGPRTase) are activated by Mg(II), Mn(II), and Co(II), and we have defined the mechanism by which these activations proceed [Biochemistry 22, 3419-3424 (1983)]. A more extensive survey of the kinds of metal ions that will activate the HGPRTase catalysis now has been completed through the use of an HPLC assay procedure. Although Fe(I1) and Ca(I1) are unable to activate this reaction, a significant activation was achieved with the addition of spectroscopically pure Zn(I1) to the assay solution. In addition some IMP synthesis resulted from the addition of Ni(I1) to the assay mixture. Both the Zn(I1) and Ni(I1) kinetic effects on HGPRTase over a limited metal ion concentration range have been analyzed through the use of curve-fitting exercises. These results, in addition to the similar pH profiles for the activations by Mg(II), Mn(II), Co(II), and Zn(II), suggest that all of these metal ions activate the HGPRTase-catalyzed synthesis of IMP by way of the same mechanism [model II as defined by London and Steck, Biochemistry 8, 1767-1779 (1969)], during which two divalent ions bind to the HGPRTase active site per molecule of PRibPP.
ABBREVIATIONS PRibPP, 5phosphoribosyl a-1-pyrophosphate; HGPRTase, hypoxanthinelguanine phosphoribosyltransferase; OPRTase, orotate phosphoribosyltransferase; PPase, inorganic pyrophosphatase; HPLC, high pressure liquid chromatography.
* This work was supported by NIH grants AM-20183 and RR-08168 (MBRS), and by awards from the CUNY Research Foundation. Address reprint requests to Dr. Donald L. Sloan, Department of Chemistry and Ph.D. Program in Biochemistry, The City College of the City University of New York, 138th Street at Convent Avenue, New York, NY 10031. Journal of Inorganic Biochemistry 28,407-415 (1986) 0 1986 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
407 0162-0134/86/$3.50
408
L. Z. Ali and D. L. Sloan
INTRODUCTION Hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) catalyzes the formation of either inosine monophosphate (IMP) or guanosine monophosphate (GMP), respectively, from phosphoribosyl pyrophosphate (PRibPP) and hypoxanthine or guanine [Eqs. (1) and (2)]. The kinetic mechanism of this enzyme isolated from yeast has been determined to be Ordered Bi Bi [l] and, as illustrated by Eqs. (3) and (4), the mechanism through which the enzyme is activated by magnesium ion’ has been elucidated 121. hypoxanthine + PRibPP = IMP + PPi, (1) guanine + PRibPP PRibPP(A) E+M
* GMP + PPi,
* (2)
+ M ’- M-PRibPP(MA),
(3)
MEM ..+Kz “2 ME Km,lc-\ MEMA-tproducts. K.s,\‘ /’ KI MEA /
(4)
Interestingly, we have observed that Co(II) and Mn(II), as well as Mg(II), activate the HGPRTase-catalyzed reactions [2], and we reasoned that other divalent metal ions might also serve as essential activators. In this paper we report the results of our examination of the effects of addition of Zn(II), Ni(II), Ca(II), or Fe(I1) to the enzyme assay solution. These studies were facilitated by the use of a previously designed HPLC assay procedure that allows us to determine if the substrates or products are being removed from the assay solution via complexation with metal ions, in addition to defining the rate of product formation [2]. MATERIALS
AND METHODS
Materials Baker’s yeast (Budweiser Brand) was obtained from Valente Yeast Inc. (Flushing, were NY). PRibPP (sodium salt), hypoxanthine, IMP, GMP, and triethanolamine supplied by Sigma Chemical (St. Louis). Chelex-100 ion exchange resin was purchased from Bio Rad Laboratories, whereas nickel chloride was supplied by Alfa Division, Ventrom Corp. (Danvers, MA). All other chemicals, including the chloride salts of zinc, iron, copper, manganese, magnesium, calcium, and cobalt were analytical grade. Methanol (Mallencroft reagent grade) was distilled prior to use during HPLC, and a Gelman “Water I” purifier was employed to prepare HPLCgrade water. Enzyme
Purification HGPRTase was isolated from yeast to electrophoretic previously published procedures [ 1, 31.
homogeneity
through the use of
’ The nomenclature for association and dissociation constants that was defined by London and Steck [Biochemistry 8, 1767-1779 (1969)] has been adapted for use in this report.
For example,
KO is an
association constant for the PRibPP-metal ion interaction [Eq. (3)] and is equal to [M-PRibPP]/ [PRibPP][M]. The parameter I&, is the dissociation constant for this interaction and is equal to the reciprocal of KO. Other binding events, defined by association constants K,, K,,, , KS, K,, K2 are illustrated in Eq. (4).
PHOSPHORIBOSYLTRANSFERASE
High-Performance
Liquid
ACTIVATION
409
Chromatography
A Waters Analytical HPLC Instrument equipped with a Model M45 solvent delivery system, model U6K sample injector, model 440 absorbance detector, and a Houston Omniscribe Recorder was employed in the enzyme assay procedure. A single 4 mm x 30 cm Waters uBondapak C1s column (equilibrated with 10% methanol) was placed on-line with the solvent delivery system at a flow rate of 1 ml/min. Samples (10 pL), from solutions containing the enzyme, substrates, and the appropriate concentration of one of the divalent metal ions, were injected into the column with a Hamilton 801 microliter syringe and eluted under 700-1OOO psi. Hypoxanthine and IMP in the eluent were detected at 254 nm with a 0.1 recorder setting. All of the solvents used in the chromatographic procedure were eluted by vacuum filtration through a 0.45-pm HA Millipore filter. Enzyme
Assay Procedure Measurements of the HGPRTase catalyzed reactions, and the activation of these reactions by increasing concentrations of divalent metal ions, were accomplished by using a modification of the method described by Flaks [3]. The assay solution consists of 0.2 ml hypoxanthine (54 PM final concentration), 0.4 ml of a PRibPP solution (100 PM final concentration), 0.1 ml of one of the standard divalent metal ion solutions, 0.1 ml of 20 mM triethanolamine (pH 7.8), and water to a final volume of 2.4 ml. The mixture was placed in a 38’ C water bath and the reaction was initiated by the addition of 1.6 ml of a dilute enzyme solution (approximately 1 milliunit). 2 Aliquots of this solution were removed at appropriate time intervals, and the reaction occurring in the samples was terminated by heating in a boiling water bath for 2 min. The samples were then clarified, first by centrifugation and then by passage through a 0.45-pm HA Millipore filter prior to the HPLC injection. The HPLC elution profiles (Fig. 1) were employed to determine the time course of the reaction as described previously [4, 51. During the kinetic analysis various concentrations of PRibPP and pH values between 6.0 and 9.5 were employed. These concentrations and pH values are listed in the text and figure legends. To ensure that no metal ion contaminents were present in the assay solutions prior to the addition of the appropriate divalent metal ion, all of the substrate and enzyme solutions, as well as the triethanolamine buffer, were eluted through Chelex- 100 minicolumns.
RESULTS
AND DISCUSSION
Survey of Activation
by Various
Metal Ions
The ability of Co(I1) to activate the synthesis of purine nucleotides [2] but not the OPRTase-catalyzed synthesis of orotidine monophosphate [7] prompted us to ask if other metal ions could serve as the essential activator of the synthesis of IMP. The results of a survey of some physiologically important metal ions are listed in Table I. As shown in Table 1, solutions of Ca(II) and Fe(I1) do not serve as activators of the HGPRTase-catalyzed reaction whereas some activation is provided by the addition of Ni(I1) to the assay solution. Interestingly, Zn(I1) possesses the ability to activate the enzymatic synthesis of IMP to the same extent as Mn(I1) and Co(I1). To our knowledge, this marks the first time that Zn(I1) has been observed to activate a 2 One unit of HGPRTase activity is defined as 1 pmole IMP formed/min.
410
L. Z. Ali and D. L. Sloan
I
IMP
I
I
II /j II
64
I hypoxanthine
I
20
Elution
Time
(min.)
FIGURE 1. HPLC assay procedure for the HGPRTase catalyzed synthesis of IMP. The initial reactant concentrations for this analysis were 54 pm hypoxanthine, 167 pm PRibPP, and 80 pm Zn(I1). The reaction conditions and HPLC elution conditions were described in the Methods
section. phosphoribosyltransferase. HGPRTase is reminiscent [S], which requires three examine the activation of Activation
by Divalent
This less specific requirement for metal ions exhibited by of the activator requirements of inorganic pyrophosphatase metal ions at the active site. Therefore, we elected to HGPRTase by Ni(II) and Zn(II) in more detail.
Nickel Ion Solutions
The addition of spectroscopically pure Ni(II) to an assay solution containing reactants except a metal ion activator leads to a relatively slow but significant TABLE 1. Observed
Optimal Velocity (V) and Optimal Metal Ion Concentration ([Ml) for Activation of the Hypoxanthine/Guanine Phosphoribosyltransferase Catalyzed Synthesis of IMP Metal ion
V
CallI) Co(H)
0.7
100”
0.4 0.7 0. I 0.7
700-I 100 100-600 400-1000 X0”
w
[Ml
Fc(II) Mg(II) Mn(I1) Ni(I1) Zn(I1)
a At metal ion concentrations reactant precipitation occurred
higher than the value shown, in the assay solution.
all of the synthesis
PHOSPHORIBOSYLTRANSFERASE
I 4-
I
I
ACTIVATION
411
I
A
A-
/_
1
10
lo2
lo3
NM Ni (II)
I 1
1
2
[MM Mg(ll)PRibPP]-’
FIGURE 2. (A) Initial velocity (u) measurements of Ni(II) activation (-•-) and Mg(II) activation (a) of the HGPRTasecatalyzed formation of IMP using the HPLC assay procedure. The concentrations of hypoxanthine and PRibP were 50 and 167 pm, respectively. (B) Double reciprocal plots of v I vs. the calculated reciprocal concentration of Mg(II)-PRibPP in the presence (-0-) and absence (-•-) of 200 pm Ni(I1). The reaction conditions and HPLC elution conditions were described in the Methods section.
of IMP (Fig. 2). The addition of concentrations of Ni(I1) higher than 0.8 mM resulted in a decrease in soluble hypoxanthine, as determined by a decrease in the HPLC elution peak amplitude of the filtered assay solution. In order to test whether this activation by Ni(I1) proceeds through a mechanism similar to the activation by Mg(I1) [2], a competition assay procedure was devised. As shown in Figure 2, a plot of the rate of IMP formation versus the calculated concentration (Z&, = 260 PM) of Mg(II)PRibPP [2], in the presence and absence of 0.2 mM Ni(II), defines a competition between the metal ions. This result suggests that the activation by Ni(I1) of the HGPRTase-catalyzed reaction proceeds through the mechanism shown in equation (4) and is defined by the kinetic equation below [Eq. (5)]. Theoretical curve-fitting exercises, making use of equation (4), were then employed to evaluate the appropriate affinity constants for the Ni(I1) activation (Table 2). The mechanism described by Eqs. (3) and (4) defines three general regions for a plot of Vversus [Ml: (1) an activation region where V increases with [Ml, (2) a region where V is independent of [Ml, and (3) an inhibition region where I/ decreases with increasing [Ml. For the activation by Ni(II), the only region that was observed was the activation region, and therefore KS and K,,, could not be evaluated.
v/ v,,, =
KcK, [Mlf [M-PRibPP] K, [Mlf (KS[PRibPP] + Km [M]f+ Kc [M-PRibPP]
+ 1) + 1
(5)
412
L. Z. Ali and D. L. Sloan
TABLE 2. Calculated
Constants for the Metal Ion Activation of the HypoxanthineBuanine Phosphoribosyltransferase Catalyzed Synthesis of IMP.”
Mn(II) Mg(II) Co(I1) Zn(I1) Ni(I1)
17 260 17 150’ 20’
22 19 22 -_d _d
1,100 900 1,100 -._d _d
6 21 5 20 20
8 11 8 50 10
0.1 3.2 0.1 15 0.4
6.2 1.50 6.2 290 10
y The definitions of these micromolar dissociation constants have been defined in a footnote to the text and by London and Steck [lo]. b The calculated parameters for Mn(II), Mg(II), and Ca(I1) and the methods for the complete error analysis of these interactions have been described previously by Ali and Sloan [2]. c These values of & were estimated from competitive effects of these metal ions on the paramagnetic effect of Mn(II) on the water proton longitudinal relaxation rates of PRibPP solutions. as defined by Victor. LeoMensah, and Sloan [7]. d Only the inhibitory region of the initial velocity-versus-[M(II)] curve can be used to define the value of these parameters, and since this region was not characterized for these metal ions, the values of I?, and x,,, used in the curve-fitting exercises were those determined for Mg(I1).
Activation
by Divalent
Zinc Ion Solutions
Activation of the enzyme-catalyzed formation of IMP by Zn(I1) was examined at three different PRibPP concentrations and a range of Zn(I1) concentrations. The rate decreases that were observed at concentrations beyond 100 ,wM were attributed to a loss of soluble base in the assay mixtures prior to the initiation of the reaction, since these decreases also occurred in assay solutions that contained no PRibPP. However, the concentration of hypoxanthine remained unchanged when the range of S-100 PM Zn(I1) was employed in the assay procedure, and theoretical curves that employed equation (5) were generated to describe these results (Fig. 3). The constants that resulted from these calculations have been compiled in Tables 1 and 2. These results reveal that Zn(I1) is an effective activator of the HGPRTase-catalyzed reaction, over this limited metal ion concentration range.
I
I
I / I I , /I
I
/
, , ( , I ,(
6-
” x10
1
10
MM Znlll)
lo2
FIGURE
3. Initial velocity (u) measurements of the Zn(I1) activation of the HPIGPRTase-catalyzed formation of IMP using the HPLC assay procedure. The concentration of hypoxanthine in the incubation solutions was 54 pm. The concentrations of PRibPP employed were 33 pm (-•-), 67 pm (-0-). and 167 /*rn (-0-). The reaction conditions and HPLC elution conditions were described in the Methods section.
PHOSPHORIBOSYLTRANSFERASE
ACTIVATION
413
pH Analysis The final question we wished to answer concerned the pH effect on the various metal ion activations. As shown in Figure 4, the pH where each of the activations is maximal is within the same range (pH 8) whereas at pH 9, Co(I1) and Zn(II), but not Mn(I1) or Mg(II), have lost the ability to activate this reaction. [In contrast to this enzymatic reaction, the optimum pH at which Zn(II) activates inorganic pyrophosphatase, pH 6, is much lower than the optimum pH values observed for Mn(II), Mg(II), and Co(II).] These results suggest that the mechanism by which each of these metal ions activates the enzymatic synthesis of IMP is the same. Moreover, the results do not reflect coordination of the metal ions with hypoxanthine, since the crossover coordination of Ni(I1) and Zn(I1) with the N-7 or N-9 positions of this base occurs at pH values of 7.1 and 6.7, respectively [9], and this coordination would inhibit nucleotide formation., Mechanism
by Which
HGPRTase
is Activated
by Metal Ions
The activation by a variety of divalent metal ions has been examined for two other yeast enzymes, orotate phosphoribosyltransferase (OPRTase) and inorganic pyrophosphatase (PPase) [7, 81. Although the reactions catalyzed by PPase and HGPRTase are quite different, the mechanisms by which they are activated are apparently similar, since the obligatory active form of PPase is also an M2E complex. Not surprisingly, both enzymes proceed with a broad metal ion specificity. In contrast, the OPRTasecatalyzed reaction, which is analogous to those catalyzed by HGPRTase, proceeds
FIGURE 4. The pH profiles for the activations of the HGPRTase-catalyzed synthesis of IMP by Mn(I1) (-0-), Zn(I1) (-A-), Co(U) (-A-), and Mg(II) (-•-) in the presence of 170 PM PRibPP. The concentration of each metal ion was maintained at 83 PM. The reaction conditions as well as the HPLC elution conditions were as described in the Methods section.
V
x10
I
I
I
1
7
8
9
PH
J
414 L. Z. Ali and D. L. Sloan
through the use of a Ping Pong Bi Bi kinetic mechanism and is activated by metal ions via a mechanism that provides a role for two metal ions but requires only one essential metal ion [7]. This activation is defined as Model III by London and Steck [lo], in which both the apo-enzyme (E) and holo-enzyme (ME) forms of OPRTase are active [Eq. (6)]. The common factor in the mechanisms by which HGPRTase and OPRTase are activated [Eqs. (4) and (6), respectively] is the requirement for a metal ionPRibPP complex (MA) as the true substrate [2. 71. ME = MEMA &t ES EMA
-+ products -+ products
(6)
If indeed the metal ions coordinate only with the enzymes and with the phosphate groups of PRibPP, then several mechanisms by which HGPRTase may utilize two metal ions to catalyze the formation of a nucleotide can be envisioned. During one such mechanism, the enzyme-bound metal ion would coordinate the 5 ‘-phosphate group and position it away from the ribose ring so that on-line displacements at the 1 ‘position would be facilitated. During an alternate mechanism, the two metal ions are positioned on opposite sides of the pyrophosphate group and not only neutralize in part the negative charges present on the pyrophosphate portion of PRibPP, but form an ionic bond with the negative charge that would result from an on-line pyrophosphate displacement by hypoxanthine. Both of these mechanisms would be consistent with the observation that high Mg(I1) and Mn(I1) concentrations, where ME. MzE, and MzPRibPP complexes would exist, are inhibitory [2]. The order in which transition metal ions coordinate ligands. the Irving-Williams order of stability constants [ 11, 12, 131, is Ni(I1) > Zn(I1) > Co(H) > Mn(II), whereas the order of affinity of metal ions for HGPRTase-bound PRibPP (Table 2) is estimated to be Co(H) = Mn(I1) > Zn(I1) = Ni(I1). This unusual order indicates that the coordination of these metal ions at the HGPRTase active site is to both amino acid residues and to the phosphate and pyrophosphate groups of PRibPP. Chemical modification studies of the essential amino acids of HGPRTase underway currently may reveal which residues coordinate these essential metal ions.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
L. Z. Ali and D. L. Sloan, J. Biol. Chem. 257, 1149-l 155 (1982). L. Z. Ali and D. L. Sloan, Biochemistry 22, 3419-3424 (1983). R. Schmidt, H. Weigand, and U. Reichert, Eur. J. Biochem. 77, 77-85 (1979). J. G. Flaks, Methods Enzymoi. 6, 144-148 (1963). D. L. Sloan, Adv. Chromatog. 23, 97-125 (1984). D. L. Sloan, L. Z. Ali, D. Aybar-Bat&a, C. Yan, and S. L. Hess. J. Chromatog. 316, 43-50 (1984). J. Victor, A. Leo-Mensah, and D. L. Sloan, Biochemistry 18, 3597-3604 (1979). K. M. Welsh, A. Jacobyansky, B. Springs. and B. S. Cooperman, Biochemistry 22, 2243-2248 (1983). R. B. Martin, Act. Chem. Res. 18, 32-38 (1985). W. P. London and T. L. Steck, Biochemistry 8, 1767-1779 (1969). H. Irving and R. J. P. Williams, J. Chem. Sot., 3192 (1953).
PHOSPHORIBOSYLTRANSFERASE
ACTIVATION
415
12. M. N. Hughes, Inorganic Chemistry of Biological Processes, Wiley, London, 1972, Chap. 3, p. 71. 13. L. G. Sillen and A. E. Martell, Stability Constants of Metal-Ion Complexes, The Chemical Society, London, 1964.
Received June 30, 1986; accepted July 9, 1986