t78

Biochimica et Biophysica Acta, 1122 (1992) 178- ! 82 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34243

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production Rafael Radi ~', Sidhartha Tan b, Eugenio Prodanov a, Roy A. Evans c and Dale A. Parks c.d a Department of Biochemistry, Faculty of Medicine, Unicersity of the Republic, Montecideo (Uruguay), t, Department of Pediatrics, Unicersity of Alabama at Birmingham, Birmbzgham, AL (USA), " Department of Anesthesiology, Unicersity of Alabama at Birmbzgham, Birmingham, AL (USA) and ,t Department of Physiology, Unicersity of Alabama at Birmingham, Birmingham, AL (USA) (Received 20 November 1991)

Key words: Xanthine oxidase; Uric acid; Xanthine; Superoxide; Free radical; Kinetics

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 ~M xanthine and 210 ILM oxygen, the apparent K i for uric acid was 70 ~M. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enz3,me-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.

Introduction

Xanthine oxidase (xanthine :oxygen oxidoreductase EC 1.2.3.2) is a complex metalloflavoenzyme which participates in reactions of purine and possibly xenobiotic metabolism [1,2]. Xanthine oxidase has a broad specificity towards electron donors, including many different heterocyclic molecules and aldehydes [1]. Still, it is accepted that its primary function in vivo is to oxidize hypoxanthine and xanthine into uric acid, using molecular oxygen as electron acceptor. In xanthine oxidase-catalyzed reactions, oxygen can be reduced by one or two electrons giving rise to superoxide ( 0 2) or hydrogen peroxide (H202) , respectively [3-5]. The basic catalytic mechanism of xanthine oxidase reactions can be ascribed to as a ping-pong cycle, with the electron donor binding in the first place to the active site [1]. Xanthine oxidase has four redox centers in the active site, which participate in electron transfer reactions. They are molybdenum (Mo), two iron-sulfur clus-

Correspondence: D.A. Parks, Department of Anesthesiology, University of Alabama at Birmingham, UAB Station, Birmingham, AL 35294, USA.

ters and the flavin adenine dinucleotide center (FAD) [1]. The molybdenum and F A D can hold up to two electrons ea~ h; the iron-sulfur clusters can accept only one electron. Thus, xanthine oxidase can bear a total of six electrons in the active site under strongly reducing conditions, The different redox centers are thought to be in a fast equilibrium [6], and normally intramolecular electron transfer at the xanthine oxidase active site occurs from the molybdenum to the FAD center, the sites of entry and egress of reducing equivalents, respectively [1]. The iron sulfur centers serve as electron carriers between the Mo and FAD sites [7]. Percentages of divalent and monovalent electron flux to oxygen are extremely variable depending on the experimental conditions. The factors affecting monovalent to divalent ratio include nature and concentration of the substrate, pO 2 and pH [3,8,9]. In general, factors that lead to a decreased level of enzyme reduction, will favor the univalent flux ( 0 2 production). For instance, reduction of oxygen under 50/~M xanthine, at pH 7.4 and 25°C, is performed 75% through divalent flux (1202) and 25% through monovalent flux ( 0 2) [3]. Product inhibition is observed in different enzymatic systems and inhibition patterns vary depending on the inhibitory mechanism [10]. Xanthine oxidase is inhibited at high concentrations of hypoxanthine and xan-

179 thine [8,11,12]. The reaction product, uric acid, could also be an inhibitor, because its chemical structure differs minimally from that of the substrates. Although it has been previously reported [13], inhibition of xanthine oxidase by uric acid has not been comprehensively studied and there are no reports on the effects of uric acid on the reoxidation pathways of the enzyme. In this investigation, we performed steady-state kinetic studies to address the mechanisms of xanthine oxidase inhibition by uric acid and the influence of product inhibition on mono and divalent electron flux to oxygen. Materials and Methods

Hypoxanthine, xanthine, horse heart cytochrome c (type VI) were purchased from Sigma (St. Louis, MO). Bovine erythrocyte superoxide dismutase (SOD) was obtained from Griinenthal (Germany) and xanthine oxidase (1.2 U/mg) from Calbiochem. All other reagents were of analytical grade. Spectrophotometric measurements were carried out on a Gilford Response II spectrophotometer. Initial rates of uric acid production were calculated from the increase in absorbance at 292 nm (e = 12 raM-~ cm-~, Ref. 8) and initial rates of superoxide production were calculated from the SOD-inhibitable reduction of cytochrome c at 550 nm (e = 21 mM-~ cm-m, Ref. 14). Reactions were camed out in either 50 mM potassium phosphate (pH 7.4) or 50 mM sodium carbonate (pH 9.5). Temperature of the assays was 25°C in all cases. Data reported are the average of three to five experiments that were performed in triplicates on separate days.

Results

Inhibition of xanthine oxidase by uric acid. Steady state expc~hnents were performed under nonsaturating concentrations of substrates. Uric acid inhibition was studied with respect to the purine substrate xanthine (4 to 12/zM) and oxygen was kept constant at 210/zM in all studies. The results of these experiments carried out in 50 mM potassium phosphate, pH 7.4 at 25°C are shown in Fig. 1. Uric acid (0 to 65 /~M) was an uncompetitive inhibitor with respect to the reducing substrate as seen in the Lineweaver Burk plot (Fig. 1A) where the lines at different concentrations of uric acid are parallel. The corresponding primary Dixon plot confirms the uncompetitive characteristic of xanthine oxidase inhibitor by urate and shows a linear inhibition pattern (Fig. 1B). A linear inhibition pattern (Fig. 1B) indicates that no more than one molecule of uric acid per molecule of enzyme was needed to cause inhibition. Primary Dixon plots do not allow determination of K~ values in uncompetitive type inhibition [10]. Thus, we applied a secondary Dixon plot which plots the y axis intercept of Fig. 1A vs. the concentration of uric acid, which indicated an apparent K i of 65 p,M. Inhibition type and K i values were independently confirmed applying the plot of Cornish-Bowden (Fig. 3, Ref. 15). In this plot, a K~ of 70/zM was determined by taking the median of all intercepts. Analogous steady state experiments performed in 50 mM sodium bicarbonate (pH 9.5) also indicated an uncompetitive inhibition by uric acid with an apparent K~ of 250 /zM, which is in agreement with the less pronounced inhibition of xanthine oxidase by purines at high pH [1,8].

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L_ 0

I 25

[Uric Acid]

I 50

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Fig. 1. Sleady slale analysis of the inhibition of xanlhine oxidase by uric acid. (A) Xanthine (4 Io 12/~M) was exposed Io 1.5 muU/mi xanthine oxidase in lhe presence of 0 (o), 10/~M (z~), 25/~M ( n ) , 35 # M (I), 50/~M ( A ) or 65/~M ( I ) uric acid. Oxygen concenlralion was 210/~M. The reactions were carried out in 20 mM potassium phosphate, pH 7.4 at 25°C. (B) Same experimental data as in (A) but analyzed in the form of a Dixon plot. Symbols in this plot represent xanthine concentrations of 4/zM (o), 6 p.M ( • ), 8 gM (o). 10 p.M ( zx) and 12 gM ([]).

I80 TABLE I

Influence of product inhibition on superoxide radical production Reaction mixtures contained different critical concentrations of xanthine (X) and uric acid (UA), 1.5 mU/mi xanthine oxidase in 50 ~M potassium phosphate, pH 7.4 at 25°C. [X] (tzM)

[UA] (ttM)

Of (tzM/min)

UA (/zM/min)

Of/UA

Univ. flux (%)

6 6 6 6 6 8 8 8 8 8 12 12 12 12 12

0 10 35 50 65 0 10 35 50 65 0 10 35 50 65

0.57 0.53 0.51 0.48 0.50 0.59 0.56 0.54 0.51 0.51 0.60 0.58 0.55 0.53 0.51

0.81 0.71 0.59 0.54 0.51 0.93 0.81 0.67 0.61 0.58 1.03 0.89 0.72 0.67 0.61

0.70 0.75 0.86 0.88 0.98 0.63 0.69 0.81 0.84 0.88 0.58 0.65 0.76 0.79 0.84

35 38 43 44 49 32 35 40 42 44 29 33 38 40 42

[8,16]. Since uric acid can also bind to the reduced Mo, xanthine oxidase product inhibition could be explained by a similar mechanism involving the formation of a non-productive reduced enzyme-uric acid complex. However, uric acid inhibition could be also explained by simply considering the classical ping-pong cycle of xanthine oxidase-catalyzed reactions, initially proposed by Gutfreund and Sturtevant [17]. As the step of xanthine oxidation to uric acid is reversible, inhibition is expected at high concentrations of urate just by reversion of that step. These two potential inhibitory mechanisms are represented in the following reaction schemes, which we will call 'reversible ping-pong mechanism' and 'dead-end product inhibition'.

Reversible ping-pong mechanism EXH 2

E~"k-,

H2 0 ~

k-:x-,~E H

_4

k_ 3 ~ ~ O 2

2

k 4 X , ~ E H 2 0 2 / ~ - / k3

Influence of product inhibition on 0 2 production. Uric acid caused a concentration-dependent increase on univalent flux percentage at both pH 7.4 and 9.5 while xanthine oxidation rates were decreased (Table I). Thus, during product inhibition of xanthine oxidase, absolute rates of 0 2 production decreased proportionally less than uric acid production rate. A much stronger influence of uric acid on univalent flux was observed at pH 7.4 (Table I) than 9.5 (data not shown) which correlates with the more potent inhibition of uric acid at lower pH.

where E represents the free enzyme, EXH 2 the oxidized enzyme-xanthine complex, EH 2 the reduced enzyme, E H 2 0 2 the reduced enzyme-oxygen complex, where X H 2 is xanthine, P is uric acid and H 2 0 2 is hydrogen peroxide.

Dead-end product inhibition __EXH2

XH~," • EH2P

Discussion

H202 ~ - 4

k-~o~

k 4 X ' ~ E H 2 0 j j 2 " : /~3

This study shows that uric acid is an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate xanthine, with an apparent K i of 70/.I.M under 50 /zM xanthine and 210 /zM oxygen. This product inhibition pattern was the same as found for the xanthine analog, 8-bromoxanthine which was an uncompetitive inhibitor with a K i of 460/z M at pH 8.5 [16]. Also, the steady state equation recently derived for substrate inhibition of xanthine oxidase, also corresponds to a uncompetitive inhibition because apparent Vm and K m were the Vm and K m of a Michaelian scheme multiplied by the same factor [8,10]. Both 8bromoxanthine and substrate inhibition of xanthine oxidase appear to be due to binding of these purines to the reduced form of the Mo center of the enzyme, which results in the inability of the enzyme to participate in the reductive half reaction of the catalytic cycle

k-5 "

In this model there is participation of a new molecular species, EH 2P, representing the reduced enzyme-urate inhibitory complex. Steady state rate equations for both models were derived by the King-Altman method [18]. For the ping-pong model the equation of initial velocity with added u r a t e ( H 2 0 2 = 0, urate ~ 0) is: Vm'XH 2 1+ V= XH 2 +

o~ ~ +.~,

)

~ o, K s K m. . . . p + Ks Ksp 0 2 1+

1+

(1)

181 For the 'dead-end inhibition' model the equation of initial velocity with added urate is:

1.4

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Vm'XH 2 K°-" P i

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7

(2)

::k

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>-

0

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-50

[Uric Acid]

-K.

k4(k-t + k2) K~= kl(k 2.1.k4)

'opp

k 2 ( k - 3 + k4) K°:=

k3(k 2 + k 4)

The kinetic parameters in Eqn. 2 are defined in the same manner as for Eqn. 1. Both equations are similar and for uraite = 0 they become the .identical equation for initial velocities (Vin): Vm"XH 2 K I ~ °"

l+-~ O2 r~

(3)

o"-----7Km1+-02

Both models support product inhibition but of a mixed type, not strictly uncompetitive, because although Vm a n d K m are multiplied by the same factor in each e q u a t i o n (Eqns. 1 and 2), the expression for the appare n t K m also includes an addend that does not appear in the expression for apparent Vm. This apparent discrepancy between the models and the experimental results may be explained by considering that in the numerator of the expression for apparent K m, K~ is significantly greater than the product to which it is added. S

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Vin = - XH-, +

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Vm

/O o