Indian 10urnal of Biochemistry & Biophysics Vol. 39, February 2002, pp. 55-59

Inactivation of maize NADP-malic enzyme by Cu2+-ascorbate

S E Pinto, S R Rao* and A S Bhagwat

Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India

Received 29 May 2001; revised 26 December 2001

Maize malic enzyme was rapidly inactivated by micromolar concentrations of cupric nitrate in the presence of ascorbate at pH, 5.0. Ascorbate or Cu2+ alone had no effect on enzyme activity . The substrate L-malate or NADP individually provided almost total protection against Cu2+-ascorbate inactivation. The loss of enzyme activity was accompanied by cleavage of the enzyme. The cleaved peptides showed molecular mass of 55 kDa, 48 kDa, 38 kDa, and 14 kDa. Addition o f EDTA, hi stidine and imidazole provided protection . The results of protection experiments with sodium azide, DABCO and catalase suggested that reactive oxygen species were generated resulting in loss of enzyme activity. This was further supported by experiments showing that the rate of enzyme inactivation was higher in 0 20 than in water. It is suggested that maize malic enzyme is modified by reactive oxygen species like singlet oxygen and H20 2 generated by Cu2+_ ascorbate system and the modified amino acid residue(s) may be located at or near the substrate-binding site of the enzyme.

Maize NAOP malic enzyme [L-malate: NAOP oxidoreductase (decarboxylating) , EC 1.1.1. 40] catalyzes the oxidative decarboxylation of malate to pyruvate in presence of divalent metal ions. In C4 plants, the enzyme is located in bundle sheath chloroplasts and CO2 released by oxidative decarboxylation of malate is refixed by ribulose-l,5-bisphosphate carboxylase/oxygenase. Malic enzyme has been found in most living organisms, including bacteria, yeast, plants and humans and other animals and has highly conserved amino acid sequences. Chemical modifications of the maize malic enzyme had implicated histidyll , arginyl2, cysteinye, carboxyl4, and ty rosyl5 residues involved in the catalysis by the enzyme. We had earlier reported6 that ASp352 was the coordination site for Mg2+ binding in maiz~ malic enzyme when Fe2+-ascorbate at neutral pH Vi as used as metal catalysed ox idation (MCO). ASp352 is a conserved residue in mal ic enzyme from all sources and its role in metal bindi ng was confirmed from crystallograph ic data of human mitochondrial malic enzyme7

.

A number of MCO systems cause the inactivation f 68· 11 A . MCO o enzymes' '. non-enzymatic ~ystem

comprises ascorbate, O2 and Fe (III) / Fe (II) or Cu+ / Cu2

+. MCO of either Asp, His, Cys, Met, Trp, and Tyl at the catalytic site of a number of enzymes has been

* Author for correspondence Fax: +91225505151 E-mail : sudhar@apsara. barco ernel.in Abbreviations used: MCO, metal calalyzed ox idation: DABCO, I ,4-diazabicyclo-(2.2.2.) octane: SOD, superox ide dismutasc.

reported6.s.lO. The inactivation mechanism involves

the generation of reactive oxygen species in the presence of reduced transition metals such as Fe2

+ and Cu+. Oxidation of these metal ions by molecular oxygen yields superoxide radical anion, which undergoes dismutation reaction to form hydrogen peroxide. Hydrogen peroxide, in turn, can react with reduced metal ions via the Fenton reaction, generating hydroxyl radical. Reducing equivalents such as ascorbate and OTT are needed to recycle the oxidized metal ions .

Studies on pigeon liver malic enzyme with different MCO (Fe2

+ and Cu2+) systems at neutral and acidic pH indicated that MCO systems at different pH show different specificities in protein modifications and peptide bond cleavage. ln addition to ASp258 (ASp352 of maize malic enzyme) as metal binding ligand (identified by Fe2+-ascorbate) at neutral pH, three additional aspartate (ASp I4 1, ASp194, and ASp4M) were identified by Cu2+ -ascorbate system at acidic pH as the coordination sites for the metal binding in pigeon liver malic enzyme 13

.

In the present study we have compared the inactivation of maize ma lic enzyme at neutral and acidic pH by Fe2

+ -ascorbate and Cu2+ -ascorbate

systems. We had reported previously the inactivation mechanism of maize mal ic enzyme and identified A 352 M?+ b' d' . b . F 2+ b sp as g- . m mg site y usmg e -ascor ate at neutral pH. The present paper attempts to study the mechanism of inactivation of maize malic enzyme by Cu2+-ascorbate at acidic pH (PH 5) and also to check for addi tional coordination si tes for metal binding in

56 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 39, FEBRUARY 2002

the maize NADP-malic enzyme, as seen for the pigeon liver malic enzyme.

Materials and Methods

Chemicals Most of the biochemicals like NADP, malate and

catalase were obtained from Sigma Chemical Co., USA. SOD was obtained from Boehringer Mannheim India. All other chemicals including cupric nitrate were of analytical reagent grade.

Enzyme preparation, assay and protein estimation NADP-malic enzyme from maize leaves was

purified to apparent homogeneity according to method described by Asami et al. 14

• The final sp. activity of the enzyme was 70 units per mg protein per min . One unit was defined as the amount of enzyme that catalyses an initial I Ilmole of NADPH formation per min under assay conditions A molecular mass of 250 kDa was used for the calculation of enzyme protein concentrations. Malic enzyme was assayed in a I-ml reaction mixture containing 50 mM Tris-HCl (PH 7.5), 5 mM malate, 0.23 mM NADP, 10 mM MgCI2 and appropriate amount of enzyme. The formation of NADPH at 25°C was monitored at 340 nm with a UVICON-940 spectrophotometer (Kontron instruments, Japan). Protein concentration was determined according to the method of Asami et al. (1979).

Enzyme inactivation The enzyme (1-2 11M) in 50 mM sodium-acetate

(PH, 5.0) or 50 mM Tris-HCI (PH, 7.5) treated with the indicated concentrations of freshly prepared cupric nitrate and 20 mM ascorbate. Inactivation was monitored by assaying the enzyme activity in small aliquots withdrawn at different time intervals and quenched with 4 mM EDTA. Controls (with only ascorbate or with copper without ascorbate) were run. Other experimental conditions are provided in the respective Table or Figure legend.

SDS-PAGE The native and the inactivated proteins were run on

SDS-PAGE using the method of Laemmli 15 (Stacking ge l, 7%; Separating gel, 12%.). Separation was carried out with a constant current of 60 rnA on a Techno source (India) electrophoresis system. Molecular masses of the fragments of malic enzyme were estimated using standard protein markers (Amersham Pharmacia Biotech).

Results and Discussion

Inactivation of maize malic enzyme at neutral and acidic pH by Fe2

+ -ascorbate and Cu2+ -ascorbate

. Fig. 1 (A and B) shows the inactivation of NADP-I· f . . h F 2+ d C 2+ rna IC enzyme rom maIze WIt e, an u-

ascorbate system at neutral pH (Tris-HCI, pH 7.5) and acidic pH (sodium acetate, pH 5.0) respectively . In both systems at neutral pH, the inactivation was faster than at acidic pH. The time dependence of inactivation at acidic pH was almost similar in both systems.

Inactivation at varying concentrations of Cu2+ and

20 mM ascorbate at acidic pH Incubation of maize malic enzyme with micromolar

concentrations of Cu2+ in presence of 20 mM

ascorbate at acidic pH resulted in a rapid and irreversible loss of enzyme activity (Fig. 2). The time course of inactivation did not follow pseudo-first­order reaction kinetics. The presence of ascorbate, oxygen and Cu2

+ in the reaction mixture was essential for inactivation as reported in the case of several other enzymesS

•13

.16

• Presence of ascorbate or Cu2+ alone

had no effect. EDT A (4 mM) provided complete protection against Cu2

+ -ascorbate inactivation of malic enzyme (Table 1).

Protection of NADP-malic enzyme against inactivation by Cu2

+ -ascorbate Effect of varying concentrations of substrate and

co-factor on the rate of enzyme inactivation by Cu2+_

ascorbate was examined. Preincubation of enzyme with varying concentrations of eithe malate or NADP provided substantial protection (Table 2) agai nst

100 2+ 2+ '" \ (B) Cu c Fe \

c 80 , .. , E 60 • ' ... ..

0:: ,

"'''',,, '--.. ~ ~ 40 v

' 1' >

-.""~ ~"-;; 20 ;l. ~"

10 20 30 {,O 0 10 20 30 ~o

Tim.lminl

Fig. I-Inactivation of maize NADP-malic enzyme by (A): Fe2+ or (8) Cu2+-ascorbate at neutral and acidi c pH [Maize NADP­malic enzyme (1-2 f!M) in 50 mM sodium acetate (PH 5.0. . ----. . .... ----.... ). or in 50 mM Tris-HCI (PH 7.5,0----0, L'.----L'.); in presence of 20 ).1M ferrous sulphate plus 20 mM sodium ascorbate, (. , 0) or 20 ).1M cupric nilrate plus 20 mM sodium ascorbate ( .... , L'.) at 25°C. At the indicated time intervals a small aliquot removed and used for assaying enzyme activity after termination of reaction with 4mM EDT A

PINTO el at.: INACTIVATION OF MAIZE ~MDP-MALIC ENZYME 57

100

01 80 c:

. f;

" E 60 '" a: >.

:~ 40 u <l: ;;! 20 ~

0 0 10 20 30 40

Time (min)

Fig. 2-Time course of inactivation of the malic enzyme for varying Cu2+ concentration in presence of 20 mM sodium ascorbate [The reaction mixture contained (1-2 ~M malic enzyme in 50 mM sodium-acetate, pH 5.0) . At the indicated times aliquots were removed and assayed as stated in Fig. I. Cu2+ concentrations were (~----~); 5 ~M; ( .... ----.... ); 10 ~M; (0----0); 15 ~M;

(e----e ); 20 ~Ml

Table I-Effect of active oxygen scavengers, EDTA, DABCO, imidazole and histidine on the inactivation of malic enzyme by

Cu2+ -ascorbate

[The enzyme (1-2 /lM) was preincubated with catalase 18.7 /lg/mrl (sp. activity, 10.9 unit I mg solid) or SOD, 10 /lglmr l (sp. activity 5000 unitlmg), or n-propyl gallate, or DAB CO, or sodium azide, or imidazole or histidine or EDT A in 50 mM sodium acetate buffer (PH 5.0) for 10 min and then with 10 /lM Cu2+ plus 20 mM ascorbate for 60 min]

Ligands % Activity remaining

None I DO.O

Cu2+ (0.0 I mM) 10.0

Catalase 67 .7

SOD 10.0

n-propyl gallate (I mM) 8.0

Sodium azide (I mM) 80.0

DABCO (I mM) 85 .0

EDTA (4 mM) I DO.O

Cu2+ (0.01 mM) 20.0

Imidazole (2 .5mM) 74.0

(5 .0 mM) 84.0

(10.0 mM) 87.0

Histidine (10 mM) 72.0

Cu2+-ascorbate induced inactivation. Malate and NADP alone at 0.5 mM and 1 mM respectively, provided maximum protection. Malate (0.5 mM) plus Mg2+ (l0 mM) resulted in complete protection of malic enzyme activity (Table 2). These results indicated that the residue involved in modification

Table 2-lnactivation of maize NADP-malic enzyme by Cu2+ -ascorbate and protection by substrate

[The enzyme (1-2 /lM in 50 mM acetate buffer pH 5.0)) was preincubated with different ligands for 10 min. and treated with 10 /lM Cu2+ plus 20 mM ascorbate for 40 min . A small aliquot (10 /ll) was removed and assayed for enzyme ac tivity after termination of the reac tion with 4 mM EDTAj

Addition

None

Cu2+

Malate

NADP

Malate + Mg2+

NADP+Mg2+

Concentration (mM)

0.010

0.010

0.025

0.050

0.250

0.500

0.010

0.Q25

0.050

0.100

0.250

1.000

0.500 + 10

0.250 + 10

% Activity remaining

100.0

21.0

60.0

70.0

72.4

75.0

89.6

69 .5

73.3

75.8

80.1

82.9

84.5

99.8

53.6

may be located at the active site, probably at the substrate binding site. Earlier, Chou et al. t3 observed only 15% protection by malate (0.5 mM). NADP (0.23 mM) did not provide any protection against Cu2+ -ascorbate.

SDS-PAGE pattern of Cu2+ ascorbate inactivated

NADP-malic enzyme The inactivated malic enzyme (with residual

activity 1 %) along with native (uninactivated) protein was subjected to SDS-PAGE to study the oxidative cleavage of the protein. The native malic enzyme is a tetra mer with a subunit molecular mass of 62 kDa. The inactivated protein (99% loss of enzyme activity) was cleaved into four fragments of 55 kDa, 48 kDa, 38 kDa, and 14 kDa and this resulted in the considerable reduction of 62 kDa band of modified protein by Cu2+-ascorbate. Since cleavage fragments gave very diffused bands on PAGE and could not be obtained in sufficient quantity , sequence analysis was not carried out.

EJrect of dijferent oxygen scavengers on the inactivation of malic enzyme

Effect of active oxygen scavengers (Table 1) on the loss of malic enzyme activity by Cu2+ -ascorbate was

58 INDIAN 1. BIOCHEM. BIOPHYS. , VOL. 39. FEBRUARY 2002

100 OJ C c 80 ro E Q.)

60 a: i?:' .:;

40 U « ;;!! 20

0 0

• • • • •

20 40 60 80 100 120

Catalase [)l91

Fig. 3-Effect of different concentrations of catalase (sp. acti vity, 10.9 units/mg) on the inactivation of maize malic enzyme at 10 /AM C1I 2

+ plus 20 mM ascorbate. [The enzyme (1-2 /AM in 50 111M sodium acetate buffer, pH 5.0) was preincllbated with different concentrations of catalase and treated with Cu2

+ plus ascorbate for 60 min . The enzyme ac ti vity was measured as stated in Fig. I]

100 OJ C

. ~ 80 ell E & 60 i?:' .:; 40 :0 . « ~ 0

20

0 0 10 20 30 40 50

Time [min]

Fig. 4-Effect of 0 20 on Cu2+ induced inactivation of maize

malic enzyme at 10 /AM Cu2+ plus 20 mM ascorbate [The reaction

mixture contained malic enzyme (1-2 p.M in 50 mM sodium acetate buffer, pH S.D). e----e, H20 ; 0----0, 0 20 . The enzyme activity was measured as stated in Fig. 1]

Table 3-lnactivation of maize NAOP- malic enzyme by H2 O2

[The enzyme (1-2 p.M, in 50 mM sodium acetate buffer pH 5.0) was treated with H20 2 for 60 min. An aliquot (10 p.1) was used for measuring the enzyme activity]

Addition Concentration (mM) % Activity remaining

0.25

0.50

1.00

100.0

17.0

8.9

8.6

studied. Catalase (for hydrogen peroxide), superoxide dismutase (for superoxide anion), n-propyl gallate (for hydroxyl and alkoxyl radicals I7

) , sodium azide and OABCO (for singlet oxygen l 8

) were used as scavengers for various active oxygen species.

Catalase protected 68% enzyme actIVIty, while OABCO and sodium azide (both I ruM) provided 80% and 85 % protection respectively . These results confirmed that the inacti vation of malic enzyme by Cu2+ -ascorbate was predominantly due to the generation of reactive oxygen species (Hz0 2 and si nglet oxygen) in the reaction mixture.

The effect of different concentrations of catalase on the inactivation of malic enzyme is shown in Fig. 3. Catalase at 20 f.!g/ml provided 70% protection and there was near total protection above 50 JL g/ml. Protection of malic enzyme by catalase confirmed the formation of H20 2 in the reaction mixture during inactivation of enzyme by Cu2+-ascorbate.

Addition of 250 f.!M H20 z to the reaction mixture I'esulted in 83% loss of enzyme activity in 60 min (Table 3). Kim et al. 9 observed that catalase protected yeast glutamine synthetase from inactivation by Fe2+_ ascorbate while H20 2 alone, at high concentration (10 ruM) for 2 hr, caused no protein cleavage but inactivated the enzyme partially .

As seen in Table 1, OABCO and sodium azide also offered protection against Cu2+-induced inacti vation of malic enzyme activity. The rate of singlet oxygen dependent reactions is known to increase in 0 20 as singlet oxygen has longer lifetime in 0 20

19.2°. The

loss of malic enzyme activity due to Cu2+ -ascorbate inactivation was found to be about 20% faster in 0 20 than in water (Fig. 4), confirming the generation of singlet oxygen in the reaction mixture.

Histidine can chelate metal ions21 or can act as scavenger for singlet oxygen22

• The effects of histidine and imidazole on the inactivation of malic enzyme activity by Cu2+ -ascorbate were studied, (Table 1). It can be seen that histidine at 10 mM reduced the loss of enzyme activity by about 70%. Addition of varying concentrations of imidazole to the reaction mixture also protected malic enzyme activity against Cu2+-ascorbate inactivation (Table 1). In our earlier studies6 with the Fe2+-ascorbate system, protection of maize malic enzyme activity by imidazole was not found.

References 1 lawa1i N & Bhagwat A S (1987) Phytochemistry 26, 1859-

1862

2 Rao S R, Kamath B G & Bhagwat A S ( 1991 ) Phytochemistry 30, 431-435

3 Orincovich M F & Andreo C S (1994) Biochim BioplJys Acta 1206,10-16

4 Rao S R, Kamath B G & Bhagwat A S (1997) Indian J Biochem Biophys 34, 253-258

PINTO et al.: INACTIVATION OF MAIZE NADP-MALIC ENZYME 59

5 Rao S R, Kamath B G & Bhagwat A S (1999) Photosynthetica 36, 225-231

6 Rao S R, Kamath B G & Bhagwat A S (1998) Plant Physiol Biochem 36, 799-804

7 Xu Y, Bhargawa G, Wu H, Loeber G & Tong L (1999) Structure, 7, 877-R84

8 Levine R L (1983) J Bioi Chem, 258, 11823-11827 9 Kim K, Rhee S G & Stadtman E R (1985) J Bioi Chem 29,

15394-15397 10 Farber 1 M & Levine R L (1986) J Biol Chem 261 , 4574 -

4578 II Sounder S & Colman R F (1993) J Biol Chem 268, 5264 -

5271 12 Wei C H, Chou W Y, Huang S M, Lin C & Chang G G

(1994) Biochemistry 33, 7931-7936 13 Chou W Y, Tsai W P, Lin C C & Chang G G (1995) J Biol

Chem, 270, 25935-25941

14 Asami S, Inoue K, Matsumoto K, Murachi A & Akazawa T (1979) Arch Biochem Biophys 194, 503-510

15 Laemmli U K (1970) Nature (London) 227, 680-685 16 Amici A, Levine R L, Tsai L & Stadtman E R (1989) J Bioi

Chen1264,3341-3346 17 Bors N, Langebartels C, Michel C & Sandermann H lr

(1989) Phytochemistry 28, 1589- 1595 18 Foote C S (1976) in Free Radicals in Biology (Pryor W A

ed), 2, 85-133, Academic Press New York 19 Merkel P B, Nilsson R & Kearns D R (1972) J Am Chem

Soc, 94, (I), 1030-1031 20 Nilson R & Kearns D ( 1975) Photochem Photobiol 17. 65-68 21 Fucci L Oliver C N, Coon M 1 & Stadtman, E R. (1983) Proc

Natl Acad Sci (USA) 80, 1521-1525 22 Davison A 1, Kettle A 1 & Fatur D J (1986) J Bioi Chem 261 ,

1193 -1200.