ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 343, No. 1, July 1, pp. 146–148, 1997 Article No. BB970129
RESEARCH REPORT Transient Kinetics of Polyamine Oxidase from Zea mays L. Andrea Bellelli,* Riccardo Angelini,1 Maria Laurenzi, and Rodolfo Federico *CNR, Centro di Biologia Molecolare, Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli,’’ Universita` degli Studi di Roma ‘‘La Sapienza’’; and Dipartimento di Biologia, Universita` degli Studi ‘‘Roma III,’’ Rome, Italy
Received February 27, 1997, and in revised form April 2, 1997
Transient kinetics of pure polyamine oxidase from Zea mays L. has been analyzed by stopped flow and a simple catalytic mechanism has been hypothesized; the rate constants for each step of the catalytic cycle have been determined and the calculated values for the steady-state conditions agree with the independently determined experimental values. The enzyme reacts rapidly with spermidine (one of its physiological substrates) and has a low Km (S), suggesting that the oxidation of the amine in vivo is quite efficient; on the other hand, the rate constant for the reaction with oxygen is low and is responsible for the high value of Km (O2). Despite the ubiquitous occurrence of aliphatic polyamines in prokaryotic and eukaryotic organisms and their implication in growth and key developmental processes (1, 2), poor attention has been devoted to the study of polyamine catabolism and only recently the biochemical properties and the physiological modulations of the enzymes involved in this process have been studied in detail (3–5). Vertebrate polyamine oxidases participate in the interconversion pathway of polyamine degradation and transform spermidine and spermine, and more efficiently their N1-acetyl derivatives, into putrescine and spermidine, respectively, plus propanal or acetamidopropanal and H2O2 (1, 5). In plant (4), bacteria (1), and protozoa (6), 1,3-diaminopropane, 4-aminobutyral, or 3-aminopropyl-4-aminobutyral and H2O2 are the reaction products of spermidine and spermine oxidation catalyzed by polyamine oxidases analyzed to date. As these compounds cannot be converted again to other polyamines, polyamine oxidase is considered to be involved in terminal catabolism of polyamines in these taxa. Moreover, N1-acetylspermine, which is the best substrate for vertebrate polyamine oxidases, acts apparently as a noncompetitive inhibitor for maize and oat enzymes (7). The plant enzymes, which apparently occur only in the monocots, have been purified and partially characterized from few species (4). It has been suggested that these enzymes may have a role in the production of H2O2 in the
1
To whom correspondence should be addressed at Dipartimento di Biologia, Universita` degli Studi ‘‘Roma III,’’ Viale G. Marconi 446, I-00146 Rome, Italy. Fax: Int /6 55 176 321. E-mail:
[email protected].
cell wall, needed for lignification, wall stiffening, and defense (8). Maize polyamine oxidase, the most studied plant member of this enzyme class, is a monomeric glycoprotein with a Mr of 53 1 103 containing one molecule of FAD2; the sugar content of the enzyme is 2.5%, mainly represented by arabinose (9). Plant polyamine oxidase generally shows higher catalytic activity than do those from animals and their absorption spectrum in the visible range makes it suitable for a detailed analysis of the reaction mechanism (9). When oxidized maize polyamine oxidase is anaerobically mixed with its substrate spermidine, a large decrease in absorbance at 450 nm is recorded (this wavelength corresponds to the maximum extinction of the oxidized polyamine oxidase), consistent with the reduction of the flavin cofactor (9); if the concentration of spermidine is high enough to sustain a steady-state condition, the cofactor is reduced to approximately 50% (Fig. 1). The end point of the reaction depends on which substrate is in excess: thus, if oxygen exceeds spermidine, the enzyme ends up in the oxidized state (Eox), while if the opposite condition holds the reduced form (Ered) is the final species. The data of Fig. 1 were analyzed using a nonlinear least-squares minimization routine developed in our laboratory using the Borland Pascal compiler. Several kinetic schemes were examined, but even the simplest one (Scheme 1) offers a satisfactory description of the experimental data, with the assumption that three species of the enzyme are populated in the catalytic cycle, two of which share the same extinction, i.e., the oxidized enzyme Eox and the Michaelis complex ES, while the reduced enzyme (Ered) is colorless. Under the experimental condition of Fig. 1 the absorbance of species Eox and ES has been assigned the value of 0.04 in agreement with extinction coefficient of 7000 M01 cm01 for FAD (10), while that of Ered has been assigned the value of 0.001. 1,2
4
The algebraic formulas employed to derive the steady-state parameters are as follows: 2
Abbreviation used: FAD, Flavin adenine dinucleotide.
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Eox / S / O2 } ES / O2 r Ered / P / O2 r Eox / P [1]
0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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KINETICS OF PLANT POLYAMINE OXIDASE
FIG. 1. Steady-state kinetics of 5 mM maize polyamine oxidase at fixed oxygen concentration (270 mM after mixing) and variable concentration of spermidine (open squares, 500 mM; solid squares, 150 mM; open circles, 50 mM; solid circles, 15 mM; triangles, 5 mM, after mixing). Stopped-flow experiments were carried out using an Applied Photophysics MV17 apparatus, recording the optical changes at 450 nm; the optical path is 1 cm. The enzyme and spermidine solution were air equilibrated, buffer was 0.1 M potassium phosphate, temperature was 257C. Polyamine oxidase was purified as previously described from shoots of 10-day-old maize seedlings grown at 257C in the dark (8).
fixed [S] Å X;
kcat(O2) Å k1rXrk3/(k2 / k3 / k1X) Km(O2) Å kcat(O2)/k4
fixed [O2] Å air
kcat(S) Å k4[O2]k3/(k4[O2] / k3) Km(S) Å k4[O2](k2 / k3)/k1(k4[O2] / k3).
The parameter search for such a simple scheme is relatively rapid, in spite of the fact that the differential kinetic equations require numerical integration (carried out by the Runge Kutta four-order algorithm) (11), and allows an easy estimate of the steady state parameters, as reported in Table I. It will be noticed that steady-state parameters are calculated for each substrate on the assumption of a fixed concen-
TABLE I
Rate Constants and Steady-State Parameters for the Reaction of Maize Polyamine Oxidase with Spermidine and Oxygen, as described by Scheme 1 3.3 1 106 1 s01 183 s01 6.3 1 105 27 mM 88 s01 198 mM 125 s01
k1 k2 k3 k4 Km (S) kcat (S) Km (O2) Kcat (O2)
01
s01
01
s01
M
M
Note. Km (S) is defined as the concentration of spermidine required to obtain V Å 0.5 1 Vmax in air-equilibrated solutions, while Km (O2) is the concentration of oxygen required to achieve the same result at a fixed concentration of spermidine (100 mM); kcat is the turnover number under the same conditions.
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FIG. 2. Pre-steady-state kinetics of polyamine oxidase. Solid symbols, reaction of 10 mM oxidized maize polyamine oxidase with 50 mM spermidine in the absence of oxygen (both solutions were evacuated using a vacuum pump and equilibrated with 1 atm of pure nitrogen); open symbols, reaction of 10 mM reduced maize polyamine oxidase (in the presence of excess spermidine and equilibrated under 1 atm N2) with air-equilibrated buffer ([O2] 135 mM after mixing). All concentrations are after mixing; other experimental conditions as in Fig. 1.
tration of the other (270 mM for oxygen and 100 mM for spermidine). The kinetic parameters obtained from this analysis are reported in Table I; the values of Km(S) and kcat(S) calculated from the kinetic constants agree with those obtained from steady-state experiments carried out at 0.2 nM polyamine oxidase [Km(S) Å 22 mM and kcat(S) Å 90 s01] (9). The reaction of the oxidized enzyme with excess spermidine can easily be explored in the absence of oxygen (polyamine oxidase in carefully degassed buffer, solid symbols in Fig. 2) to obtain an independent estimate of k1; this experiment, reported in Fig. 2, yielded a pseudo-first-order time course for the bleaching reaction and allowed us to calculate a second-order rate constant of 5.0 1 106 M01 s01, in acceptable agreement with that obtained in the experiment presented in Fig. 1 (notice that the enzyme concentration of 10 mM after mixing in the experiment reported in Fig. 2 is twice the value of that in Fig. 1). Figure 2 also shows the time course recorded upon mixing reduced polyamine oxidase (i.e., the enzyme equilibrated with nitrogen in the presence of excess substrate, open symbols) with air-equilibrated buffer; the end point of this reaction is a steady-state mixture and its apparent rate constant at 23 s01 offers only a very rough approximation of the value of k4 (approximately one-third of the value reported in Table I). The catalytic mechanism employed to describe the results obtained in this work is a minimal one and several other intermediates may be populated to a small extent in the cycle. Even with this limitation, the analysis of the data reported above reveals some interesting characteristic of maize polyamine oxidase, the most surprising being perhaps the low rate constant for the reaction with oxygen (in comparison to those of the other reactions), responsible for the high value of Km (O2) (198 mM, Table 1). The direct measure of the reoxidation of the enzyme (Fig. 2, open symbols) confirms this finding. A consequence of this kinetic feature is that maize polyamine oxidase is only partially oxidized even under fully aerobic
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conditions and that oxygen concentration may be a relevant rate-limiting factor in vivo. On the other hand, the reaction with spermidine is much more efficient and both the turnover number and the low Km suggest that the transformation of the amine proceeds rapidly under physiological conditions. On the basis of their reactivity with oxygen and the reaction they catalyze, flavoproteins may be grouped into four classes (10): the flavine dehydrogenases that react slowly with oxygen to give mainly H2O2 but also some O0 2 ; the electron transferases that react rapidly with oxygen producing mainly O0 2 and the flavoprotein neutral radical; the oxidases that react rapidly with oxygen with the production of oxidized flavoprotein and H2O2; and finally the monooxygenases that catalyze the splitting of the oxygen bonds, inserting one oxygen atom into a substrate and reducing the other atom to H2O. The exceptionally wide range of the rate constants for oxidation of flavin observed in different flavoenzymes has been ascribed to the protein environment in which the reduced flavin is located (10). In this context maize polyamine oxidase shows a somewhat slower reactivity versus oxygen than glucose oxidase (k Å 2.2 1 106 M01s01) (10), but has the otherwise typical second-order reaction which does not populate intermediate species (e.g., flavin semiquinone). It is also instructive to compare the value of Km (O2) and k4 of maize polyamine oxidase with the corresponding values of 360 mM and 2.9 1 104 M01 s01 obtained for the couple beef liver monoamine oxidase B/benzylamine (12); this enzyme is noteworthy because the rate constant describing its oxidation depends on the amine substrate used, which forces one of at least two alternative kinetic mechanism. That a similar condition may also apply to maize polyamine oxidase is an uncertain but stimulating hypothesis.
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ACKNOWLEDGMENTS This research was supported by ‘‘Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica-Progetti Nazionali M.U.R.S.T. 40%.’’ This paper is dedicated to Alessia Paglia, prematurely deceased on January 10, 1997.
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