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A steady-state kinetic investigation of the uricase reaction mechanism
ARCHIVES
OF BIOCHEMISTRY
AND
A Steady-State
BIOPHYSICS
Kinetic
163, 359-366 (19’i4)
Investigation
of the Uricase
Reaction
Mechanism’ 0. M. PITTS Department
of Biochemistry,
Medical
D. G. PRIEST
AND
University
of South Carolina,
Received January
Charleston,
South Carolina
29401
1’7, 19i4
The steady-state kinetics of porcine liver uricase has been investigated over an extensive range of both mate and oxygen concentration. Spectrophotometric assay of the accumulation of reaction intermediate which absorbs strongly in the ultraviolet region as well as the oxygen electrode were used to minimize inaccuracies in initial velocity measurements. The superoxide radical could not be detected as a product of the uricase reaction. Reciprocal plots with both urate and oxygen showed extensive nonlinearity. Mechanisms involving second binding sites on uricase appear unlikely in view of the structural properties of the enzyme. A random substrate binding scheme has been shown to be consistent with all of the nonlinear reciprocal plots and is thus suggested as the most probable mechanism of action for uricase.
The steady-state kinetic mechanism of uricase has been investigated by Baum et al. (1). They proposed an ordered sequential binding of the substrates oxygen and urate based on the observation that the apparent K, and V for urate are different in air and in a 100% oxygen atmosphere. A more extensive investigation of the oxygen dependence of initial velocity was conducted by Davidson (2); however, a single urate concentration was used for these studies. Under the conditions of his experiments initial velocity did not describe a rectangular hyperbola when plotted vs oxygen tension. The type of behavior shown has been termed “substrate activation.” Apparent high substrate inhibition by urate reported by Baum et al. (1) has subsequently been shown to be caused by spectrophotometric assay error introduced by the accumulation of an intermediate that absorbs strongly in the same uv region as urate (3, 4). Such assay difficulties can be overcome by measuring accumulation of the intermediate spectrophotometrically or
by use of the oxygen electrode. A detailed steady-state kinetic investigation using these assay methods has yielded reciprocal plots for both urate and oxygen which are extensively nonlinear. A mechanism consistent with the nonlinear reciprocal plots and with the structural properties of uricase is proposed. MATERIALS
AND
METHODS
Porcine liver uricase was obtained from Boehringer-Mannheim Corporation. These preparations had a specific activity of 8.0 IU per mg protein and were used without further purification (51. Uric acid was obtained from Calbiochem, San Diego, CA. Urate solutions were prepared in glycine buffers on the day of use. A molar extinction coefficient at 290 nm of I.23 x 10’ cm-l was used to establish concentration (6). Other chemicals were obtained from Fisher Scientific Company. Oxygen-nitrogen mixtures ranging from 5% to 80’; oxygen were obtained from Matheson Gas Products, Atlanta, Ga. For spectrophotometric studies at oxygen tensions other than atmospheric these mixtures were bubbled through substrate solutions in Thunberg cuvettes for 12 min. This was more than sufficient time to attain complete experimental equilibration. The solubility of oxygen at 20% saturation was ‘Acknowledgement is made to the Research Cmassumed to yield 240 pM solutions (3). Concentrations poration and to the 1972 South Carolina State Approat other oxygen tensions were calculated proportionpriation for Research for support of this research. ally. 359 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
360
PITTS
AND PRIEST
Absorption spectrophotometry was conducted on an ACTA C-III double beam recording spectrophotometer with a thermostated cell chamber. A Clarktype oxygen electrode (Yellow Springs Instruments) equipped with a strip chart recorder was used to monitor oxygen depletion. Initial slopes from a minimum of six replicate assays for each substrate concentration were convertd to molecular activities, averages, and standard deviations with the use of an IBM-360 digital computer. A molecular weight of 125,000 for uricase was used (5). Solid lines on reciprocal plots were calculated by manual iteration on a DIGITAL-PDP-8 computer.
36
P
RESULTS Uricase catalyzes the oxidation of urate with molecular oxygen as the only known electron acceptor. In alkaline solution the stable products of this reaction have been shown to be allantoin, carbon dioxide, and hydrogen peroxide (4, 7-10). In high borate concentration buffer systems evidence has been presented that alloxanate and urea are the predominant final stable products of oxidized urate (10). In either case at least one unstable intermediate (8) is traversed subsequent to the enzyme-mediated oxidation of the urate moiety. While it is clear that hydrogen peroxide represents the final reduction product of oxygen (9), the possible formation of a reactive intermediate, the superoxide radical, during this process needed further investigation. The reduction of nitrobluetetrazolium as well as other methods have been used successfully to detect the superoxide radical with other enzymatic and nonenzymatic systems (11-13). Figure 1A shows conclusively that under the conditions of these experiments no nitrobluetetrazolium reduction could be detected at 0.240 or 1.200 mM oxygen even though the uricase was turning over very rapidly. Figure 1B shows that nitrobluetetrazolium reduction can be detected quite easily in the xanthine oxidase system when the enzyme is turning over at an approximately equivalent rate to that of uricase (Fig. 1A). Therefore, by the nitrobluetetrazolium reduction method, no superoxide is detected during the uricase reaction. These results were confirmed both spectroscopically using cytochrome c as the detection agent (11, 12) and by the oxygen electrode method using cytochrome
!
04
‘b
0
TIME
(MIN)
1. Absence of superoxide radical production by uricase using nitrobluetetrazolium as the detection agent. Experiments were performed according to Beauchamp and Fridovich (13). In all cases 0.1 M glycine, pH 8.8, with 1 x lo-” M EDTA was the buffer. A. Studies on the uricase system at 100 WM urate using 50 ~1 of a 0.2 mg/ml uricase solution. Rates of intermediate production were measured at 312 nm at 1.20 mM (0) and 0.24 mM (0) oxygen. Nitrobluetetrazolium (2.5 x 10m5 M) was then added in attempts to detect superoxide production at 560 nm at 1.20 mM (0) and 0.24 mM (W) oxygen. B. Rate of urate production (A) by the control xanthine oxidase system at 293 nm using 100 WM xanthine and 50 ~1 of a 1.0 mg/ml xanthine oxidase solution. (A) designates the rate of nitrobluetetrazolium reduction monitored at 560 nm using the same xanthine and xanthine oxidase concentrations. FIG.
c as the superoxide scavenger (12). Therefore, it is proposed that the following products are formed during the enzyme-catalyzed portion of the overall uricase reac-
Prior to extensive steadv-state kinetic studies with uricase it was necessary to establish the validity of the assay method used. This is esneciallv true since we have previously shown that the determination
STEADY-STATE
KINETICS
of urate disappearance at 293 nm can lead to error caused by a significant absorbance of the intermediate species at this wavelength (3, 4). In one case such error led to the erroneous conclusion that uricase undergoes high substrate inhibition (1, 4). On the other hand, determination of the TABLE
I
A COMPARISON OF MOLECULAR ACTIVITIES OF URICASE OBTAINED BY THE 31%nm SPECTROPHOTOMETRIC ASSAY AND THE OXYGEN
Substrate concentrations (mM)
Urate, 1.0 o,, 1.200 0,,0.600 0,,0.060
Oxygen, Urate, Urate, Urate, Urate, Urate,
0.240 1.0 0.100 0.040 0.01 0.005
a With standard replicates.
ELECTRODE
Mean velocity (molecular activity)” O2 Electrode
Spectrophotometric (312 nm)
2852 * 83 2016 ~47 1249 * 200
1890 * 42
1868 zt 38 1487 * 74 12Olzt 54 678 i 50 429 i61
deviations
2871i60 2010 *55 1210 * 72
1476 i61 1222 * 50 681* 74 445 zt 89
of a minimum I’-
of six
OF URICASE
361
rate of accumulation of the intermediate absorbing species can be determined at 312 nm. It can be seen in Table I that a comparison of this assay with the rate of oxygen depletion as determined by the oxygen electrode, yields essentially identical results. Thus, all subsequent kinetic studies were performed using the 312-nm assay. To obtain a better understanding of the kinetic mechanism by which uricase catalyzes the oxidation of urate an extensive investigation of initial velocity as a function of both urate and molecular oxygen concentration has been conducted. Davidson (2) has previously determined initial velocities, using manometric techniques, as a function of oxygen concentration at a single urate concentration. When these results are replotted in reciprocal form significant downward curvature at the higher oxygen concentrations can be detected. Figure 2 confirms these results and shows that such curvature at high oxygen concentration is apparent over a broad range of urate concentrations. At the higher urate concentrations apparent linearity is obtained at oxygen concentrations below 0.60 mM. As urate concentration is -7
FIG. 2. Reciprocal of molecular activity at 25°C vs reciprocal of oxygen concentration at 15,25, 40, 100, and 1000 PM urate. All assays were conducted at 312 nm with substrates in 0.1 M glycine buffer, pH 8.8. Urate solutions were equilibrated 12 min by bubbling water-saturated oxygen-nitrogen mixtures through 3.0 ml of solution in Thunberg cuvettes. Reactions were initiated with 25 ~1 of a 0.2 mg/ml uricase solution in the sidearm. Points indicate the means of a minimum of six replicate assays. Error bars are standard deviations. Lines are calculated as explained in the text.
362
PITTS
AND PRIEST
lowered, nonlinearity in the higher oxygen effect were reported to have been caused by concentration region becomes less pro- the second substrate, oxygen. It is assumed nounced and the error about each point I I I I I I. I becomes higher. When urate was used as 40t / 4 the variable substrate nonlinearity was also obtained (Fig. 3). At high urate concentrations and relatively low oxygen tensions downward curvature can be observed. This “substrate activation” is diminished as oxygen concentration is increased. In the low urate concentration region in Fig. 3 there is again a suggestion of additional downward curvature. This region was thus investigated more thoroughly. Figure 4 confirms that downward curvature is observed essentially throughout this low urate concentration region. The error is naturally much higher at such low urate concentrations. A single point at very low urate concentration has been included in Fig. 4 to indicate the limits of the detection I I I I I I method. Regardless of the increased error, 0.0’ 60 20 40 100 160 however, it is clear that significant down[URATEI x 10~~ ward curvature occurs. FIG. 3. Reciprocal of molecular activity at 25°C vs DISCUSSION
Baum et al. (1) have previously gated the steady-state kinetics of using urate as a variable substrate oxygen concentrations. Both a K,
0.01 0
investiuricase at two and V
I
20
reciprocal of urate concentration over the range 1.7 mM-0.01 mM at 0.06, 0.12, 0.24, 0.60, 0.96, and 1.20 mM oxygen concentrations. The top curve represents the lowest oxygen concentration. Points indicate means, error bars are standard deviations, and lines were calculated as explained in the test. Other conditions are the same as in Fig. 2.
I 40
II 60
FIG. 4. Reciprocal of molecular activity at 25°C vs reciprocal of urate concentration over the range 1.7 mM-0.0016 mM at 0.12, 0.24, 0.60, and 1.20 mM oxygen. The top curve represents the lowest oxygen concentration. As many as 9-12 replicates were used in the region below 15 PM urate. Other conditions are the same as in Fig. 2.
STEADY-STATE
KINETICS
that Baum et al. (1) used extrapolated lines to obtain V and K, at atmospheric 0, due to their reported marked substrate inhibition above 100 PM. Since no data or urate range was reported for the 1.2 mM 0, experiments other than the K, and V values, it is not clear if apparent substrate inhibition was observed at this 0, concentration. An ordered mechanism was suggested involving the sequential binding of the two substrates, urate and oxygen. We have observed that reciprocal plots with urate under atmospheric oxygen conditions are approximately linear over the urate concentration range used by Baum et al. (1). However, with mate concentrations outside this range, or when oxygen is used as the variable substrate, significant nonlinearity is observed in reciprocal plots. Such nonlinearity makes ordered mechanisms untenable. We have, therefore. sought other mechanisms which are consistent with such behavior of reciprocal plots. One mechanism which could give rise to such nonlinearity involves a second binding site or allosteric enzyme model. If urate or oxygen bind at multiple sites on the enzyme surface, and these sites interact in the appropriate manner, a suitable model could be obtained. A second site binding and/or the binding of more than one urate molecule at the active site was previously considered by Baum et al. (1) to explain the substrate inhibition which they observed. A second site model appears unlikely for uricase because only one copper atom per active enzyme molecule could be detected and copper in all likelihood is intimately involved in both substrate binding and electron transfer from urate to oxygen. We are thus forced to abandon allosteric mechanisms for this enzyme. Sweeny and Fisher (14) have shown that significant nonlinearity is obtained in rate equations derived from random binding schemes. Such mechanisms appear to be the most likely explanation for nonlinearity in the reciprocal plots with uricase. Fisher et al. (15) have suggested methods to approach the selection of the most likely binding schemes for enzymes whose mechanism involves complex reac-
363
OF URICASE
tion sequences. This method involves determination of a general kinetic scheme based upon possible chemical events and stoichiometry of the reaction, followed by a reduction of this general model to simpler models through agreement of steady-state kinetic data with the appropriate derived rate equations as well as through chemical or physical detection methods. We have chosen to follow such an approach with this enzyme system. Figure 5A shows the appropriate general model to be considered for uricase. This
I
-M-E
I
‘-
EU
c;
8.
E
++
-
EU
HZ02\1 EOz 02 *-
1
++ -E-h
j EUO2
1 I
I / -
EH202
I E
7
I
EU IJ
f C
E+iU. ”
FIG. 5. Single-site models for the uricase enzymic reaction. Unless otherwise designated substrates binding to or products being released from parallel steps are the same. E designates the free or unbound form of the enzyme and u designates urate. Charges do not necessarily refer to the copper oxidation state, but are to help make clear the number of electrons removed in a given step. In Fig. 5C reaction steps 2,4, 6, and 8 are the reverse of reaction steps 1, 3, 5. and 7, respectively. Steps 9 and 10 are shown as irreversible.
364
PITTS
AND PRIEST
model incorporates the sequential binding pathway previously proposed by Baum et al. (l), an alternate sequential substrate binding pathway, a “ping-pang”-type pathway, and pathways involving superoxide as a reaction product. Enzyme species generated from the superoxide pathways are connected by dashed lines. It should be noted that the superscripts on individual enzyme forms do not necessarily indicate the oxidation state of the enzyme but rather are used to indicate the number of electrons which are transferred in individual reaction steps. Deadend substrate inhibition pathways, such those proposed by Baum et al. (l), have previously been shown to be due to assay error (4) and are thus not included. The general model, Fig. 5A, can be reduced to the simpler model shown in Fig. 5B on the basis of results presented in Fig. 1. That is, superoxide radical is not detectable as a product of the uricase reaction under the conditions used to obtain nonlinear reciprocal plots. The sensitivity of the detection methods used would allow very low quantitites of this radical to be observed. We thus conclude that enzyme forms generated through the release of oxygen which has been reduced by only one electron can be eliminated for consideration. The upper, outside, “ping-pang” pathway shown in Fig. 5B is unlikely because it would require a second free enzyme form which has been oxidized by two electrons. The copper ion in biological systems typically does not attain oxidation states other than +l and +2. For the “ping-pang” pathway to be functional the original free enzyme form must bind oxygen and transfer an electron pair to the enzyme prior to leaving the surface. In this case the enzyme, and presumably the metal, would be oxidized by two electrons. If for example, Cu+ were the original oxidation state, then Cu3+ would be required for the second free enzyme form in this pathway. Although such an oxidation state has been proposed in one other case, galactose oxidase (16), it would have to be highly unstable and is assumed unlikely for uricase. The elimina-
tion of this pathway yields the binding mechanism shown in Fig. 5C. Since product-binding steps could not be investigated either through reversal of the reaction or product-inhibition studies, they are simply shown as irreversible ordered steps. This does not indicate that such a mechanism for product release is proposed, but simply that such mechanisms could not be investigated by steady-state techniques. The rate equation for model 5C in the notation of Fisher and Hoagland (1’7) is shown below. 1 -zz
Q
-
1
1
-
-
1
k, + G + (fa) i k311021 + k,[Ul
k,
’
k,k,
’
’
+ k&,[O,l + k,M,[WWl
k,
+ M,FJl ’
where
WI ! ke k, ’ k,k, fn = [%I U-J1 ke -______ kz k, + k,k, + k, + k,k, and
Kbl
--c-
f* =
k, [O,l k, -k, + k,k,
k,
k&s VI
k,
+ 12, + k,k,
All product concentrations are set equal to zero under initial velocity conditions and thus terms containing such concentrations are not shown. To test this rate equation against the initial velocity plots shown in Figs. 2-4, simulations were conducted on an IBM-360 by iteration of the pertinent rate constants (18). A set of initial rate constants was selected by this means which gave rise to substrate activation in the appropriate concentration regions. Additional simulations were then conducted on a PDP-8 DIGITAL computer to refine these rate constants to the set shown in Table II. When the rate constants shown in
STEADY-STATE
KINETICS
TABLE II RATE CONSTANTS FOR URICASE MECHANISM SHOWN IN FIG. 5C” k, k, k,
k, k, k, k,
1.0 x 10’ 3.0 x 10' 2.5 x 10’ 3.0 x 10’
1.0 x 107 1.0 x 10’
k,
1.0 x 109 3.6 x lo3
k, k 10
4.0 x 103
1.0 x 10’
* Units of all first-order constants are min I and of all second-order constants are liter x mole- ’ x min I.
OF URICASE
365
TABLE III FRACTIONAL VELOCITIES OVER THE LINEAR REGION MARKED BY ARROWS IN FIG. 3 Urate ,uM 10 15
fa
fb
0.100
0.900
20 25 40 50 70
0.122 0.143 0.163 0.217 0.250 0.308
0.878 0.857 0.837 0.783 0.750 0.692
100
0.379
0.621
thus be concluded that linearity over a restricted substrate range should not be Table II were substituted into the above used as a priori evidence for strictly orrate equation all solid lines shown in Figs. dered binding mechanisms. In summary, uricase catalyzes the trans2-4 were obtained. Care was taken that the fer of two electrons from urate to oxygen law of microreversibility was maintained within the chemical cycle. In no case was a giving rise to both an unstable intermediate and hydrogen peroxide. Detailed rate constant allowed to assume an unusually high or low value (19). Therefore, the steady-state kinetic studies reveal extenin reciprocal plots with simple random binding scheme shown in sive nonlinearity uricase. Multiple enzyme binding site Fig. 5C is concluded to be a completely mechanisms have been excluded as the satisfactory mechanism for the extreme source of this nonlinearity based on metal nonlinear kinetics exhibited by uricase. and hence implied binding During simulation studies it was noted composition stoichiometry. A simple random substrate that apparently linear regions of reciprocal plots can be obtained even though exten- binding scheme is proposed as the most sive randomness is operational. That is, a likely mechanism. This mechanism is consistent with all of the reaction products and significant amount of an alternate pathway steady-state kinetic data available for urican be in use and apparently linear reciprocal plots observed over a restricted sub- case. It has also been shown that apparent linearity in reciprocal plots over restricted strate range. A region of the line obtained substrate concentration regions is insuffiat the lowest oxygen concentration is cient to eliminate possible random binding marked by arrows in Fig. 3. This region would appear linear in the absence of mechanisms for enzymes. initial velocities obtained at higher urate concentrations. The proportional use of the ACKNOWLEDGMENTS two pathways marked by fa and fb in Fig. 5C were calculated for this region and the We are grateful to Mr. Michael May, Dr. C. F. results are presented in Table III. It can be Lam, and Ms. Nancy Schatz for assistance in the seen that linearity is maintained even computer programming required in this work. though utilization of pathway fb is reduced from 90% to approximately 62% of the total REFERENCES reaction flux. Thus, it is apparent that a 1. BAUM, H., HUBSCHER, G., AND MAHLER, H. R. significant shift from one pathway into (1956). Biochem. Biophys. Actn 22, 514. another pathway due to changing substrate 2. DAVIDSON, J. N. (1942) Biochem. J. 36, 252. concentration can occur arid reciprocal 3. PRIEST, D. G., AND PIT-B, 0. M. (1972) Anal. Bioplots remain apparently linear. It must them. 50, 195.
366
PITTS
AND PRIEST
4. Prrrs, 0. M., AND PRIEST, D. G. (1973) Biochemistry 12, 1358. 5. PITTS, 0. M., PRIEST, D. G., AND FISH, W. W. (1974) Biochemistry 13, 888. 6. KALCKAR, H. M. (1947) J. Biol. Chem. 167, 429. 7. MAHLER, H. R. (1963) in The Enzymes (Bayer, P. D., Lardy, H., and Myrblick, K., eds.), Vol. 8, p. 285, Academic Press, New York. 8. MAHLER, H. R., BAUM, H., AND HUBSCHER, G. (1956) Science 124, 705. 9. BENTLEY, R., AND NEUBERGER, A. (1952) Biochem. J. 52, 694. 10. CANNELAKIS, E. S., AND COHEN, P. P. (1955) J. BioE. Chem. 213, 385. 11. FRIDOVICH, I. (1972) Act. Chem. Res. 5, 321. 12. FRIDOVICH, I. (1970) J. Biol. Chem. 245, 4053.
13. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276. 14. SWEENY, J. R., AND FISHER, J. R. (1968) Biochemistry 7, 561. 15. FISHER, J. R., PRIEST, D. G., AND BARTON, J. R. (1972) J. The. Biol. 37, 335. 16. HAMILTON, G. A., LIBBY, R. D., AND HARTZELL, C. R. (1973) Biochem. Biophys. Res. Commun. 55, 333. 17. FISHER, J. R., AND HOAGLAND, V. D. (1968) Aduan. Biol. Med. Phys. 12, 163. 18. MAY, M. E., AND PRIEST, D. G. (unpublished results). 19. EIGEN, M., AND HAMMES, G. G. (1963) Aduan. Enzymol. 25, 1.
OF BIOCHEMISTRY
AND
A Steady-State
BIOPHYSICS
Kinetic
163, 359-366 (19’i4)
Investigation
of the Uricase
Reaction
Mechanism’ 0. M. PITTS Department
of Biochemistry,
Medical
D. G. PRIEST
AND
University
of South Carolina,
Received January
Charleston,
South Carolina
29401
1’7, 19i4
The steady-state kinetics of porcine liver uricase has been investigated over an extensive range of both mate and oxygen concentration. Spectrophotometric assay of the accumulation of reaction intermediate which absorbs strongly in the ultraviolet region as well as the oxygen electrode were used to minimize inaccuracies in initial velocity measurements. The superoxide radical could not be detected as a product of the uricase reaction. Reciprocal plots with both urate and oxygen showed extensive nonlinearity. Mechanisms involving second binding sites on uricase appear unlikely in view of the structural properties of the enzyme. A random substrate binding scheme has been shown to be consistent with all of the nonlinear reciprocal plots and is thus suggested as the most probable mechanism of action for uricase.
The steady-state kinetic mechanism of uricase has been investigated by Baum et al. (1). They proposed an ordered sequential binding of the substrates oxygen and urate based on the observation that the apparent K, and V for urate are different in air and in a 100% oxygen atmosphere. A more extensive investigation of the oxygen dependence of initial velocity was conducted by Davidson (2); however, a single urate concentration was used for these studies. Under the conditions of his experiments initial velocity did not describe a rectangular hyperbola when plotted vs oxygen tension. The type of behavior shown has been termed “substrate activation.” Apparent high substrate inhibition by urate reported by Baum et al. (1) has subsequently been shown to be caused by spectrophotometric assay error introduced by the accumulation of an intermediate that absorbs strongly in the same uv region as urate (3, 4). Such assay difficulties can be overcome by measuring accumulation of the intermediate spectrophotometrically or
by use of the oxygen electrode. A detailed steady-state kinetic investigation using these assay methods has yielded reciprocal plots for both urate and oxygen which are extensively nonlinear. A mechanism consistent with the nonlinear reciprocal plots and with the structural properties of uricase is proposed. MATERIALS
AND
METHODS
Porcine liver uricase was obtained from Boehringer-Mannheim Corporation. These preparations had a specific activity of 8.0 IU per mg protein and were used without further purification (51. Uric acid was obtained from Calbiochem, San Diego, CA. Urate solutions were prepared in glycine buffers on the day of use. A molar extinction coefficient at 290 nm of I.23 x 10’ cm-l was used to establish concentration (6). Other chemicals were obtained from Fisher Scientific Company. Oxygen-nitrogen mixtures ranging from 5% to 80’; oxygen were obtained from Matheson Gas Products, Atlanta, Ga. For spectrophotometric studies at oxygen tensions other than atmospheric these mixtures were bubbled through substrate solutions in Thunberg cuvettes for 12 min. This was more than sufficient time to attain complete experimental equilibration. The solubility of oxygen at 20% saturation was ‘Acknowledgement is made to the Research Cmassumed to yield 240 pM solutions (3). Concentrations poration and to the 1972 South Carolina State Approat other oxygen tensions were calculated proportionpriation for Research for support of this research. ally. 359 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
360
PITTS
AND PRIEST
Absorption spectrophotometry was conducted on an ACTA C-III double beam recording spectrophotometer with a thermostated cell chamber. A Clarktype oxygen electrode (Yellow Springs Instruments) equipped with a strip chart recorder was used to monitor oxygen depletion. Initial slopes from a minimum of six replicate assays for each substrate concentration were convertd to molecular activities, averages, and standard deviations with the use of an IBM-360 digital computer. A molecular weight of 125,000 for uricase was used (5). Solid lines on reciprocal plots were calculated by manual iteration on a DIGITAL-PDP-8 computer.
36
P
RESULTS Uricase catalyzes the oxidation of urate with molecular oxygen as the only known electron acceptor. In alkaline solution the stable products of this reaction have been shown to be allantoin, carbon dioxide, and hydrogen peroxide (4, 7-10). In high borate concentration buffer systems evidence has been presented that alloxanate and urea are the predominant final stable products of oxidized urate (10). In either case at least one unstable intermediate (8) is traversed subsequent to the enzyme-mediated oxidation of the urate moiety. While it is clear that hydrogen peroxide represents the final reduction product of oxygen (9), the possible formation of a reactive intermediate, the superoxide radical, during this process needed further investigation. The reduction of nitrobluetetrazolium as well as other methods have been used successfully to detect the superoxide radical with other enzymatic and nonenzymatic systems (11-13). Figure 1A shows conclusively that under the conditions of these experiments no nitrobluetetrazolium reduction could be detected at 0.240 or 1.200 mM oxygen even though the uricase was turning over very rapidly. Figure 1B shows that nitrobluetetrazolium reduction can be detected quite easily in the xanthine oxidase system when the enzyme is turning over at an approximately equivalent rate to that of uricase (Fig. 1A). Therefore, by the nitrobluetetrazolium reduction method, no superoxide is detected during the uricase reaction. These results were confirmed both spectroscopically using cytochrome c as the detection agent (11, 12) and by the oxygen electrode method using cytochrome
!
04
‘b
0
TIME
(MIN)
1. Absence of superoxide radical production by uricase using nitrobluetetrazolium as the detection agent. Experiments were performed according to Beauchamp and Fridovich (13). In all cases 0.1 M glycine, pH 8.8, with 1 x lo-” M EDTA was the buffer. A. Studies on the uricase system at 100 WM urate using 50 ~1 of a 0.2 mg/ml uricase solution. Rates of intermediate production were measured at 312 nm at 1.20 mM (0) and 0.24 mM (0) oxygen. Nitrobluetetrazolium (2.5 x 10m5 M) was then added in attempts to detect superoxide production at 560 nm at 1.20 mM (0) and 0.24 mM (W) oxygen. B. Rate of urate production (A) by the control xanthine oxidase system at 293 nm using 100 WM xanthine and 50 ~1 of a 1.0 mg/ml xanthine oxidase solution. (A) designates the rate of nitrobluetetrazolium reduction monitored at 560 nm using the same xanthine and xanthine oxidase concentrations. FIG.
c as the superoxide scavenger (12). Therefore, it is proposed that the following products are formed during the enzyme-catalyzed portion of the overall uricase reac-
Prior to extensive steadv-state kinetic studies with uricase it was necessary to establish the validity of the assay method used. This is esneciallv true since we have previously shown that the determination
STEADY-STATE
KINETICS
of urate disappearance at 293 nm can lead to error caused by a significant absorbance of the intermediate species at this wavelength (3, 4). In one case such error led to the erroneous conclusion that uricase undergoes high substrate inhibition (1, 4). On the other hand, determination of the TABLE
I
A COMPARISON OF MOLECULAR ACTIVITIES OF URICASE OBTAINED BY THE 31%nm SPECTROPHOTOMETRIC ASSAY AND THE OXYGEN
Substrate concentrations (mM)
Urate, 1.0 o,, 1.200 0,,0.600 0,,0.060
Oxygen, Urate, Urate, Urate, Urate, Urate,
0.240 1.0 0.100 0.040 0.01 0.005
a With standard replicates.
ELECTRODE
Mean velocity (molecular activity)” O2 Electrode
Spectrophotometric (312 nm)
2852 * 83 2016 ~47 1249 * 200
1890 * 42
1868 zt 38 1487 * 74 12Olzt 54 678 i 50 429 i61
deviations
2871i60 2010 *55 1210 * 72
1476 i61 1222 * 50 681* 74 445 zt 89
of a minimum I’-
of six
OF URICASE
361
rate of accumulation of the intermediate absorbing species can be determined at 312 nm. It can be seen in Table I that a comparison of this assay with the rate of oxygen depletion as determined by the oxygen electrode, yields essentially identical results. Thus, all subsequent kinetic studies were performed using the 312-nm assay. To obtain a better understanding of the kinetic mechanism by which uricase catalyzes the oxidation of urate an extensive investigation of initial velocity as a function of both urate and molecular oxygen concentration has been conducted. Davidson (2) has previously determined initial velocities, using manometric techniques, as a function of oxygen concentration at a single urate concentration. When these results are replotted in reciprocal form significant downward curvature at the higher oxygen concentrations can be detected. Figure 2 confirms these results and shows that such curvature at high oxygen concentration is apparent over a broad range of urate concentrations. At the higher urate concentrations apparent linearity is obtained at oxygen concentrations below 0.60 mM. As urate concentration is -7
FIG. 2. Reciprocal of molecular activity at 25°C vs reciprocal of oxygen concentration at 15,25, 40, 100, and 1000 PM urate. All assays were conducted at 312 nm with substrates in 0.1 M glycine buffer, pH 8.8. Urate solutions were equilibrated 12 min by bubbling water-saturated oxygen-nitrogen mixtures through 3.0 ml of solution in Thunberg cuvettes. Reactions were initiated with 25 ~1 of a 0.2 mg/ml uricase solution in the sidearm. Points indicate the means of a minimum of six replicate assays. Error bars are standard deviations. Lines are calculated as explained in the text.
362
PITTS
AND PRIEST
lowered, nonlinearity in the higher oxygen effect were reported to have been caused by concentration region becomes less pro- the second substrate, oxygen. It is assumed nounced and the error about each point I I I I I I. I becomes higher. When urate was used as 40t / 4 the variable substrate nonlinearity was also obtained (Fig. 3). At high urate concentrations and relatively low oxygen tensions downward curvature can be observed. This “substrate activation” is diminished as oxygen concentration is increased. In the low urate concentration region in Fig. 3 there is again a suggestion of additional downward curvature. This region was thus investigated more thoroughly. Figure 4 confirms that downward curvature is observed essentially throughout this low urate concentration region. The error is naturally much higher at such low urate concentrations. A single point at very low urate concentration has been included in Fig. 4 to indicate the limits of the detection I I I I I I method. Regardless of the increased error, 0.0’ 60 20 40 100 160 however, it is clear that significant down[URATEI x 10~~ ward curvature occurs. FIG. 3. Reciprocal of molecular activity at 25°C vs DISCUSSION
Baum et al. (1) have previously gated the steady-state kinetics of using urate as a variable substrate oxygen concentrations. Both a K,
0.01 0
investiuricase at two and V
I
20
reciprocal of urate concentration over the range 1.7 mM-0.01 mM at 0.06, 0.12, 0.24, 0.60, 0.96, and 1.20 mM oxygen concentrations. The top curve represents the lowest oxygen concentration. Points indicate means, error bars are standard deviations, and lines were calculated as explained in the test. Other conditions are the same as in Fig. 2.
I 40
II 60
FIG. 4. Reciprocal of molecular activity at 25°C vs reciprocal of urate concentration over the range 1.7 mM-0.0016 mM at 0.12, 0.24, 0.60, and 1.20 mM oxygen. The top curve represents the lowest oxygen concentration. As many as 9-12 replicates were used in the region below 15 PM urate. Other conditions are the same as in Fig. 2.
STEADY-STATE
KINETICS
that Baum et al. (1) used extrapolated lines to obtain V and K, at atmospheric 0, due to their reported marked substrate inhibition above 100 PM. Since no data or urate range was reported for the 1.2 mM 0, experiments other than the K, and V values, it is not clear if apparent substrate inhibition was observed at this 0, concentration. An ordered mechanism was suggested involving the sequential binding of the two substrates, urate and oxygen. We have observed that reciprocal plots with urate under atmospheric oxygen conditions are approximately linear over the urate concentration range used by Baum et al. (1). However, with mate concentrations outside this range, or when oxygen is used as the variable substrate, significant nonlinearity is observed in reciprocal plots. Such nonlinearity makes ordered mechanisms untenable. We have, therefore. sought other mechanisms which are consistent with such behavior of reciprocal plots. One mechanism which could give rise to such nonlinearity involves a second binding site or allosteric enzyme model. If urate or oxygen bind at multiple sites on the enzyme surface, and these sites interact in the appropriate manner, a suitable model could be obtained. A second site binding and/or the binding of more than one urate molecule at the active site was previously considered by Baum et al. (1) to explain the substrate inhibition which they observed. A second site model appears unlikely for uricase because only one copper atom per active enzyme molecule could be detected and copper in all likelihood is intimately involved in both substrate binding and electron transfer from urate to oxygen. We are thus forced to abandon allosteric mechanisms for this enzyme. Sweeny and Fisher (14) have shown that significant nonlinearity is obtained in rate equations derived from random binding schemes. Such mechanisms appear to be the most likely explanation for nonlinearity in the reciprocal plots with uricase. Fisher et al. (15) have suggested methods to approach the selection of the most likely binding schemes for enzymes whose mechanism involves complex reac-
363
OF URICASE
tion sequences. This method involves determination of a general kinetic scheme based upon possible chemical events and stoichiometry of the reaction, followed by a reduction of this general model to simpler models through agreement of steady-state kinetic data with the appropriate derived rate equations as well as through chemical or physical detection methods. We have chosen to follow such an approach with this enzyme system. Figure 5A shows the appropriate general model to be considered for uricase. This
I
-M-E
I
‘-
EU
c;
8.
E
++
-
EU
HZ02\1 EOz 02 *-
1
++ -E-h
j EUO2
1 I
I / -
EH202
I E
7
I
EU IJ
f C
E+iU. ”
FIG. 5. Single-site models for the uricase enzymic reaction. Unless otherwise designated substrates binding to or products being released from parallel steps are the same. E designates the free or unbound form of the enzyme and u designates urate. Charges do not necessarily refer to the copper oxidation state, but are to help make clear the number of electrons removed in a given step. In Fig. 5C reaction steps 2,4, 6, and 8 are the reverse of reaction steps 1, 3, 5. and 7, respectively. Steps 9 and 10 are shown as irreversible.
364
PITTS
AND PRIEST
model incorporates the sequential binding pathway previously proposed by Baum et al. (l), an alternate sequential substrate binding pathway, a “ping-pang”-type pathway, and pathways involving superoxide as a reaction product. Enzyme species generated from the superoxide pathways are connected by dashed lines. It should be noted that the superscripts on individual enzyme forms do not necessarily indicate the oxidation state of the enzyme but rather are used to indicate the number of electrons which are transferred in individual reaction steps. Deadend substrate inhibition pathways, such those proposed by Baum et al. (l), have previously been shown to be due to assay error (4) and are thus not included. The general model, Fig. 5A, can be reduced to the simpler model shown in Fig. 5B on the basis of results presented in Fig. 1. That is, superoxide radical is not detectable as a product of the uricase reaction under the conditions used to obtain nonlinear reciprocal plots. The sensitivity of the detection methods used would allow very low quantitites of this radical to be observed. We thus conclude that enzyme forms generated through the release of oxygen which has been reduced by only one electron can be eliminated for consideration. The upper, outside, “ping-pang” pathway shown in Fig. 5B is unlikely because it would require a second free enzyme form which has been oxidized by two electrons. The copper ion in biological systems typically does not attain oxidation states other than +l and +2. For the “ping-pang” pathway to be functional the original free enzyme form must bind oxygen and transfer an electron pair to the enzyme prior to leaving the surface. In this case the enzyme, and presumably the metal, would be oxidized by two electrons. If for example, Cu+ were the original oxidation state, then Cu3+ would be required for the second free enzyme form in this pathway. Although such an oxidation state has been proposed in one other case, galactose oxidase (16), it would have to be highly unstable and is assumed unlikely for uricase. The elimina-
tion of this pathway yields the binding mechanism shown in Fig. 5C. Since product-binding steps could not be investigated either through reversal of the reaction or product-inhibition studies, they are simply shown as irreversible ordered steps. This does not indicate that such a mechanism for product release is proposed, but simply that such mechanisms could not be investigated by steady-state techniques. The rate equation for model 5C in the notation of Fisher and Hoagland (1’7) is shown below. 1 -zz
Q
-
1
1
-
-
1
k, + G + (fa) i k311021 + k,[Ul
k,
’
k,k,
’
’
+ k&,[O,l + k,M,[WWl
k,
+ M,FJl ’
where
WI ! ke k, ’ k,k, fn = [%I U-J1 ke -______ kz k, + k,k, + k, + k,k, and
Kbl
--c-
f* =
k, [O,l k, -k, + k,k,
k,
k&s VI
k,
+ 12, + k,k,
All product concentrations are set equal to zero under initial velocity conditions and thus terms containing such concentrations are not shown. To test this rate equation against the initial velocity plots shown in Figs. 2-4, simulations were conducted on an IBM-360 by iteration of the pertinent rate constants (18). A set of initial rate constants was selected by this means which gave rise to substrate activation in the appropriate concentration regions. Additional simulations were then conducted on a PDP-8 DIGITAL computer to refine these rate constants to the set shown in Table II. When the rate constants shown in
STEADY-STATE
KINETICS
TABLE II RATE CONSTANTS FOR URICASE MECHANISM SHOWN IN FIG. 5C” k, k, k,
k, k, k, k,
1.0 x 10’ 3.0 x 10' 2.5 x 10’ 3.0 x 10’
1.0 x 107 1.0 x 10’
k,
1.0 x 109 3.6 x lo3
k, k 10
4.0 x 103
1.0 x 10’
* Units of all first-order constants are min I and of all second-order constants are liter x mole- ’ x min I.
OF URICASE
365
TABLE III FRACTIONAL VELOCITIES OVER THE LINEAR REGION MARKED BY ARROWS IN FIG. 3 Urate ,uM 10 15
fa
fb
0.100
0.900
20 25 40 50 70
0.122 0.143 0.163 0.217 0.250 0.308
0.878 0.857 0.837 0.783 0.750 0.692
100
0.379
0.621
thus be concluded that linearity over a restricted substrate range should not be Table II were substituted into the above used as a priori evidence for strictly orrate equation all solid lines shown in Figs. dered binding mechanisms. In summary, uricase catalyzes the trans2-4 were obtained. Care was taken that the fer of two electrons from urate to oxygen law of microreversibility was maintained within the chemical cycle. In no case was a giving rise to both an unstable intermediate and hydrogen peroxide. Detailed rate constant allowed to assume an unusually high or low value (19). Therefore, the steady-state kinetic studies reveal extenin reciprocal plots with simple random binding scheme shown in sive nonlinearity uricase. Multiple enzyme binding site Fig. 5C is concluded to be a completely mechanisms have been excluded as the satisfactory mechanism for the extreme source of this nonlinearity based on metal nonlinear kinetics exhibited by uricase. and hence implied binding During simulation studies it was noted composition stoichiometry. A simple random substrate that apparently linear regions of reciprocal plots can be obtained even though exten- binding scheme is proposed as the most sive randomness is operational. That is, a likely mechanism. This mechanism is consistent with all of the reaction products and significant amount of an alternate pathway steady-state kinetic data available for urican be in use and apparently linear reciprocal plots observed over a restricted sub- case. It has also been shown that apparent linearity in reciprocal plots over restricted strate range. A region of the line obtained substrate concentration regions is insuffiat the lowest oxygen concentration is cient to eliminate possible random binding marked by arrows in Fig. 3. This region would appear linear in the absence of mechanisms for enzymes. initial velocities obtained at higher urate concentrations. The proportional use of the ACKNOWLEDGMENTS two pathways marked by fa and fb in Fig. 5C were calculated for this region and the We are grateful to Mr. Michael May, Dr. C. F. results are presented in Table III. It can be Lam, and Ms. Nancy Schatz for assistance in the seen that linearity is maintained even computer programming required in this work. though utilization of pathway fb is reduced from 90% to approximately 62% of the total REFERENCES reaction flux. Thus, it is apparent that a 1. BAUM, H., HUBSCHER, G., AND MAHLER, H. R. significant shift from one pathway into (1956). Biochem. Biophys. Actn 22, 514. another pathway due to changing substrate 2. DAVIDSON, J. N. (1942) Biochem. J. 36, 252. concentration can occur arid reciprocal 3. PRIEST, D. G., AND PIT-B, 0. M. (1972) Anal. Bioplots remain apparently linear. It must them. 50, 195.
366
PITTS
AND PRIEST
4. Prrrs, 0. M., AND PRIEST, D. G. (1973) Biochemistry 12, 1358. 5. PITTS, 0. M., PRIEST, D. G., AND FISH, W. W. (1974) Biochemistry 13, 888. 6. KALCKAR, H. M. (1947) J. Biol. Chem. 167, 429. 7. MAHLER, H. R. (1963) in The Enzymes (Bayer, P. D., Lardy, H., and Myrblick, K., eds.), Vol. 8, p. 285, Academic Press, New York. 8. MAHLER, H. R., BAUM, H., AND HUBSCHER, G. (1956) Science 124, 705. 9. BENTLEY, R., AND NEUBERGER, A. (1952) Biochem. J. 52, 694. 10. CANNELAKIS, E. S., AND COHEN, P. P. (1955) J. BioE. Chem. 213, 385. 11. FRIDOVICH, I. (1972) Act. Chem. Res. 5, 321. 12. FRIDOVICH, I. (1970) J. Biol. Chem. 245, 4053.
13. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276. 14. SWEENY, J. R., AND FISHER, J. R. (1968) Biochemistry 7, 561. 15. FISHER, J. R., PRIEST, D. G., AND BARTON, J. R. (1972) J. The. Biol. 37, 335. 16. HAMILTON, G. A., LIBBY, R. D., AND HARTZELL, C. R. (1973) Biochem. Biophys. Res. Commun. 55, 333. 17. FISHER, J. R., AND HOAGLAND, V. D. (1968) Aduan. Biol. Med. Phys. 12, 163. 18. MAY, M. E., AND PRIEST, D. G. (unpublished results). 19. EIGEN, M., AND HAMMES, G. G. (1963) Aduan. Enzymol. 25, 1.