- Email: [email protected]
Involvement of lysine residue in the nucleotide binding of pigeon liver malic enzyme: modification with affinity label periodate-oxidized nadp
Inr. J. fliorhem. Vol. 14. pp. 621 to 627, 1982 Printed in Great Britain. Ail rights reserved
0020-711X/82/070621-07~3.00’0 Copyright 0 1982 Pergam~n Press Ltd
INVOLVEMENT OF LYSINE RESIDUE IN THE NUCLEOTIDE BINDING OF PIGEON LIVER MALIC ENZYME: MODIFICATION WITH AFFINITY LABEL PERIODATE-OXIDIZED NADP Department
Gu-GANG CHANG. TSU-CHUNG CHANT and TER-MEI HC’ANG of Biochemistry. National Defense Medical Center. Taipei. Taiwan. Republic (Rrceiced
16 Nocemher
of China
1981)
Abstract--l. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions. 2. The inactivation is due to the modification of an essential lysine residue. 3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification. 4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues. 5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased. 6. The binding between the modified enzyme and NADPH was impaired.
INTRODUCTION
sodium periodate. Sephadex G-10. NADP, NADPH. adcnosine Z’S’-bisphosphate, NAD. S-ADPribose, NMN. S-AMP, S-ADP, adenosine. adenine. nicotinamide, N-X-acetyl-lysine. o-glyceraldehyde (Sigma. U.S.A.); ethyleneglycol (Fischer. U.S.A.): silica gel (Camag U.S.A.);
Malic enzyme (t.-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). EC 1.1.1.40) from pigeon liver is a tetramer composed of probably identical subunits (Nevaldine er al.. 1974). Using chemical modification method, we have characterized the roles of cysteine (Chang & Hsu, 1977a; Chang & Chueh, 1980), histidine (Chang & Hsu, 1977), tyrosine (Chang & Huang, 1980), and arginine (Chang & Huang, 1981) residues in the mechanism of pigeon liver maiic enzyme. We have developed bromopyruvate as an affinity label for this enzyme (Chang & Hsu, 1973, 1977a). However, it was found that bromopyruvate actually modifies many non-essential groups outside the active centre as well. More recently. we introduced oxidized NADP as a potentially more specific aflkity label for this enzyme (Chang & Huang, 1979). In this communication, we wish to report the site and group specificities of this reagent. The results indicate that oxidized NADP is a more efficient affinity label than bromopyruvate for malic enzyme. Our results also suggest the involvement of lysine residue in the nucleotide binding of this enzyme. The half-of-the-site reactivity between nucleotide and malic enzyme is also presented.
MATERIALS
SF-5. Switzerland) were purchased from designated sources. All other chemicals were of reagent grade or better. Distilled, deionized water was used throughout this work. Pigeon liver maiic enzyme was purified according to Hsu & tardy (1967). The homogeneity of the purified enzyme was routinely checked by potyacrylamide gel efectrophoresis. The purified enzyme was dialyzed against 50 mM Tris-Cl buffer (pH 7.0 at 25’C) containing 107, glycerol and stored at -20°C. Protein concentration was determined at 278 nm. using an extinction coefficient of 0.86 for a O.ly,, (w/v) solution (Hsu & Lardy, 1967). M, 260.000 and 65.000 (Nevaldine er 01.. 1974) for the enzyme tetramer and monomer. respectively, were used for calculation of enzyme concentration.
The overall oxidative decarboxylase activity was assayed at 3o’C according to Hsu & Lardy (1967). The formation of NADPH was monitored continuously at 340nm. In some kinetic experiments. when nucleotide was used as the variable substrate, the assay was performed with a Gilford 250 spectrophotometer equipped with a IO-cm cuvette compartment. Cylindrical cuvettes with a total volume of 30 ml and IO-cm light path were used. Prepu~u&ion
AND METHODS
Mofrrials
C3H]Sodihm borohydride (Radiochemical Center England); t_-malic acid. sodium pyruvate (Calbiochem, Address for sending correspondence: Gu-gang Chang, Ph.D. Department of Biochemistry, National Defense Medical Center. P.O. Box 8244. Taipei, Taiwan, Republic of China. &C. 14 l--F
t$oxidked
NADP
Oxidation of NADP and NAD bv NafO, were nerformed essentially according to Easterbrook-Smith et’ a(.. 1976). The purity of the oxidized nucleotides was routinely checked by.t.l.cVas previously described (Chang & Huang. 1979). Radioactive oxidized NADP (6.64 mM. 5490 cpminmol) was prepared according to Bellini ef al. (1979). NADP was first treated with E3H]NaBH, (9.5 Ci/mmol) and then with periodate. The oxidized-[‘H]NADP was purified by Sephadex G-10 chromatography. Separation of oxidized621
Gu-GANG
622
[%]NADP and C3H]NaBH, was confirms by the observation of two radioactive peaks, The first half of the leading peak was pooled. The radioactivity was measured in a Packard 3320 liquid scintillation spectrometer. The purity was checked by t.1.c. (Chang & Huang, 1979). No un-oxidized nucleotide was detectable in the oxidized nucleotide preparations. Modification
ofmalic enzyme with oxidized NADP
Kinetic studies of the modification were carried out by adding freshly prepared oxidized NADP to enzyme solution in borate buffer at pH 7.5 and 24°C. Immediately after the addition of the reagent, small aliquot was withdrawn and assayed for the zero-time enzyme activity. At intervals, samples of the reaction mixture were removed and assayed for the residual enzyme activity. Further reaction of the enzyme with oxidized NADP was negligible after dilution with assay mixture containing substrate protectors. The enzyme incubated with borate buffer alone did not show any inactivation during the experimental period. The slope of the residual activity vs time semilog plot represents the observed pseudo-first order rate constant (k,,,). Protrcfion
sfudies
Protection experiments were performed as above except that the enzyme was preincubated with substrates or inhibitors for 10 min before adding oxidized NADP. The pH of substrate of inhibitor solution was adjusted to 7.5 before mixing with enzyme. The kohs of the inactivation was obtained from each experimental set. The percentage protection was catculated according to the equation:
- k,,,(protected)]/k,,,(unprotected); Identification NADP
x 100
of the amino ucid residue modified by oxidized
Malic enzyme was reacted with oxidized NADP as described above. The inactivated enzyme was reduced with a few crystals of C3H]NaBH,, dialyzed exhaustively against water, and then hydrolyzed with 6 N HCl in an evacuated. sealed ampoule at 110°C for 24 hr. The hydrolysate was subjected to t.1.c. on glass plate coated with silica gel in a solvent system of I-propanol-25% NH, (67:33, v/v) or I-butanol-acetic acid-water (60:20:20, v/v). After chromatography, the plate was dried, marks were made every 0.5 or 1 cm, and the gel of each section was removed and counted for radioactivity. The standards for the identification of the amino acid being modified were prepared from N-z-acetyl-lysine and ~-giycera~dehyde according to Dallocchio et al. (1976). Covalent
~ube~~~g qfmaiic enzyme with oxidized-[3H]NADP
CHANG er al.
was measured (Hsu & Lardy, 1967a). In the control, reagents except the enzyme were added for correction the quenching due to reagents,
all of
RESULTS malic enzyme NADP as a substrate or an inhibitor
Kinetic
properties
of
using oxidized
Oxidized NADP was previously shown to be reduced by malic enzyme (Chang & Huang, 1979). The reaction was linear with time and enzyme concentration. The apparent Michaelis constant for oxidized NADP (30 ) 2 PM) was higher than that for NADP (1 c 3 PM), with only l/10 V,,,,, of the regular assay. Oxidized NADP was shown as a non-competitive inhibitor vs t.-malate in oxidative decarboxylation. This compound also inhibited NADP reduction competitively with a ki value of 3711M (Fig. 1). indicating binding at the nucleotide site.
ln~~~i~~t~on o~malic enzyme by oxidized NADP Incubation of malic enzyme with oxidized NADP at neutral pH and 24°C resulted in a rapid loss of enzyme activity. The inactivation reaction exhibited pseudo-first order kinetics until 907,; inactivation, but the kinetics were not consistent with any simple scheme after longer-time incubation. The enzyme always retained some residual enzymatic activity after prolonged incubation. The dependence of the loss of enzyme activity on oxidized NADP concentration has been studied (Chang & Huang, 1979). A plot of kobs vs oxidized NADP concentration showed saturation behavior, and the double reciprocal plot was linear. The results suggested the formation of an enzyme-oxidized NADP binary complex before inactivation process, and oxidized NADP acts as an affinity label for malic enzyme. The structural requirement of oxidized NADP as an effective inactivator for malic enzyme was demonstrated in Fig. 2. Oxidized NAD, under the same conditions, inactivated the enzyme much slower. The kobs for oxidized NADP and oxidized NAD were 0.06 and 0.0087 min- ‘, respectively. We found that the NADcatalyzed enzyme activity was only 1.20,; that of the regular assay. However, NAD was found to be an
7
Malic enzyme (0.18 mg) was incubated with freshly prepared oxidized-[3H]NADP (0.05 _ 0.5 mM, 1.8 z 18 x 10s cpm) in 15 mM borate buffer (pH 7.5) at 24°C. Aliquot samples were removed at zero-time and after 1 hr incubation. A few crystals of NaBH, was added to stop the reaction and reduce the covalent bond. The samples were dialyzed against water to removed unbound radioactivity, and analyzed for protein-bound radioactivity. The incorporation of covalent labei was expressed as oxidized-[3HfNADP per enzyme subunit. In some experiments, malic enzyme was preincubated with NADP to protect the nucleotide site. The protected enzyme was then treated with oxidized-[sH]NADP as described above. I / C NADP
Fluorescence
1
t I.‘MI
titration
Fluorescence titration of the native or oxidized-NADPinactivated enzyme with NADPH was performed using an Aminco-Bawman spectrofluorimeter. The nucleotidc was excited at 350 nm and the emission fiuoreseence at 4.50 nm
Fig. 1. Inhibition of mahc enzyme by oxidized NADP. The enzyme activities were assayed at different levels of oxidized NADP as described in Materials and Methods with NADP as the variable substrate.
Pigeon
liver malic enzyme
623
Table 2. Inhibition
Inhibition
Analogue
0
IO
20 TIME
30
40
(man)
Fig. 2. Inactivation of malic enzyme by oxidized NADP and oxidized NAD. Malic enzyme (63pg) was incubated with 0.98 mM oxidized NADP (0) or oxidized NAD (a) in 40 mM borate buffer (pH 7.5) at 24’C. The logarithm of residual activity was plotted against time.
effective non-competitive (see next section).
inhibitor
for this
enzyme
Protection of the enzyme from inactivation by oxidized NADP Table 1 summarizes the effects of nucleotides and Full protection their analogs on inactivation. (99 f 2% in six experiments) was afforded by 8.8 mM NADP. Other nucleotides or divalent metal ions gave partial protection. Substrates malate or pyruvate alone did not give any protection, but enhanced the protective effect of lower concentration of NADP. The protection by nucleotide coenzyme strongly suggests that modification was at the nucleotide binding site. Further protective experiments were carried out to determine which portion of the NADP molecule was responsible for the protection. NAD or its analogs AMP, ADP, NMN, adenosine, nicotinamide and adenine were all found to be non-competitive inhibitors of malic enzyme (Table 2), whereas the NADP analogs adenosine 2’,5’-bisphosphate or oxidized NADP were competitive inhibitor with respect to NADP, Table 1. Effects of nucleotide the
rate
of inactivation
Addition NADP
NADPH Adenosine 2’,5’-bisphosphate NAD 5’-ADP 5’-AMP NMN Adenosine
coenzymes and inhibitors on of malic enzyme by oxidized NADP
Concentration (mM)
y0 Protection
8.8 7.0 4.5 3.7 0.9 3.7
99 92 89 79 46 86
7.0 0.9 8.8 8.8 8.8 8.8 8.8
58 46 93 90 68 79 71
Conditions were as described in Fig. 2, except that the enzyme was preincubated with the various additions listed.
of pigeon liver malic enzyme by NADP or NAD analogues
Adenosine 2’,5’-bisphosphate Oxidized NADP 5’-ADP-ribose NAD 5’-AMP 5’-ADP NMN Adenosine Adenine Nicotinamide
pattern*
Competitive Competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive
Kit (mM)
0.006 0.037: 0.6 1.1 3.8 4.2 7.1 10 33 231
* Double reciprocal plot was used to examine the inhibition pattern. t Dixon plot (Dixon & Webb, 1979) was used to evaluate the inhibition constant. $ From Fig. I. much lower Ki value than that for the NAD analogs. In examination of the structure of the inhibitors that gave protection (NADP, NADPH, adenosine 2’,5’-bisphosphate, NAD. AMP, ADP, NMN and adenosine), with the only exception of adenosine, all compounds contain a phosphoryl moiety. Nicotinamide, adenine, Pi and PP, had no effect on the rate of inactivation (data not shown). These results suggest that phosphoryl binding site was attacked by oxidized NADP. The lack of any protective effect of Pi and PP, was probably due to its inability to bind to the enzyme. The protective effect of adenosine could be due to a steric effect. and have
Identification
of the modijied amino acid
The inactivation of malic enzyme by oxidized NADP was reversible by dilution and could be made irreversible by treating the modified enzyme with NaBH, (Chang & Huang, 1979). These results strongly suggested the formation of a Schiff base between the aldehyde groups of oxidized NADP and lysyl e-amino group of the enzyme. The reversibility suggested that the inactivation was not due to a gross structural change, although local conformational change could not be ruled out. The exact nature of the amino acid being modified was identified as lysine as anticipated (Fig. 3). A modified enzyme sample was reduced with [“H]NaBH, to label the modified amino acid residue. Figure 3B shows the t.1.c. chromatogram of the acid hydrolysate developed in I-propanol: 25% NH,. The modified enzyme gave one major and one minor radioactive peaks with R, values of 0.69 and 0.84, respectively. Figure 3A shows the chromatogram of standard prepared for identification. The complete match of these two chromatograms indicates specific labeling of lysine residue by this method. However, compared to the reported values (Bellini et al., 1979; Dallocchio er al., 1976), the R, values were slightly higher in our samples probably due to different quality of the solvents. Similar results were obtained with the 1-butanol-acetic acidwater solvent system. Incubation of a SH-masked enzyme with oxidized NADP also caused inactivation (data not shown), indicating that the inactivation was
C&J-GANG CHANG C[ al
3#-OXlDIZED
MIGRATION
(cm1
Fig. 3. Thin layer chromatogram of the acid hydrolysate of oxidized-NADP-modified enzyme. Malic enzyme (0.36 mg) was incubated with 1.2mM oxidized NADP in 22mM borate buffer (pH 7.5) at 24 C for 1 hr. The inactivated enzyme (residual activity, 7”,,1 was reduced with [‘HJNaBH,, dialyzed and hydrolyzed. The standard (AI and hydrolysate (B) were chromatographied as described in Materials and Methods.
not clue to SH modification. So far lysine was the only amino acid found to be modified with oxidized nucleotides in some other enzymes (Bellini et nl., 1979; Dallocchio et al.. 1976: Easterbrook-Smith et al.. 1976). The attempts to prepare a reduced Schick base between oxidized nucleotide and arginine (Bellini rt ul.. 1979: Easterbrook-Smith et al., 1976). cysteine or histidine (Easterbrook-Smith et ul.. 1976) were not successful.
Stoichiometry of the labeling The stoichiometry was studied by incubating the malic enzyme with oxidized-[3H]NADP and reduced with NaBH,. Figure 4 shows the incorporation of oxidized-C3H]NADP to malic enzyme. A linear plot of residual activity vs [3H]incorporation gave 0.46 molecule of oxidized NADP per enzyme subunit upon extrapolation to complete inactivation. Prolonged incubation did not give further incorporation (indicated by an arrow in Fig. 4). NADP could pre-
Fig. 4. Correlation of inactivatlon and radioactivity incorporation. Malic enzyme was incubated with ouidized-[3H]NADP as described in Materials and Methods in the presence (a) or absence (0) of NADP. Enzyme activity and radioactivity incorporation were determined and calculated. The result obtained from a prolonged incubation (5 hr) was indicated by an arrow,
vent the [3H]incorporation, as well as protect the enzyme from inactivation. This result directly demonstrated the site specificity of oxidized NADP.
Figure 5 shows the effects of oxidized NADP modification on the kinetic parameters of nucleotide coenzymes. The apparent I<,,, for NADP was increased 5 _ &fold (from 1.2 + 0.3 FM to 6.4 + 0.4 FM) and that for NADPH increased Z.&fold (from 2 PM to 5.6pM) after modification. The K,,, Lalues for other substrates (malate, pyruvate) were not changed after modification (Table 3). The V,_ of the enzyme after modification was decreased (Fig. 5). These results suggest that a decrease in binding afFinity between the modified enzyme and nucleotide coenzyme was probably the cause of decreased enzyme activity. Direct evidence for the involvement of lysine residue in the nucleotide binding was provided by fluorescence titration experiments as shown in Fig. 6. Titration of the native enzyme with NADPH resulted in a binary complex formation and an enhancement of nuclcotide fluorescence.
Oxidized-NADP-modified
0 01 I /
Fig. 5. Kinetic
properties
with
0,203
CNADPHI
enzyme
04 05 ((IM-‘)
of the native and oxidized-NADP-modified enzyme. The native enzyme (2.8 pg enzyme (5.6 pg with 24”” residual activity in A and IO”:, residual activity in 8) (0) were assayed with nucleotide as the variable substrate. Velocities were calculated for the same amount of enzyme.
in A. 3.5 pg in B) (0). or oxidized-NADP-modified
8.7pg
NAOP INCORPORATED/%WNIT
failed
Pigeon Table
3. Apparent Michaelis NADP-modified Native
Substrate t-malate UADP Pyruvate YADPtI
liver malic enzyme
constants of the oxidizedmalic enzyme
enzyme
Modified
Specific* activity
K,t (PM)
Specific* activity
44 41 46 45 1.9 1.5
67 56 1.5 0.9 20 X 103 2
1.3 1.6 0.9 II 1.2 0.15
enzyme
67 66 1.3 5.5 21 x IO3 5.6
* Overall oxidative decarboxylase was assayed when t.-malatr or NADP was the variable substrate. Reductase partlnl activity was assayed when pyruvate or NADPH was the variable substrate. t Double reciprocal plot was used to evaluate the Michaelis constant.
to enhance the NADPH fluorescence, lack of complex formation.
indicating
the
DISCUSSION
In previous studies. bromopyruvate was shown to be a useful affinity label in the study of reaction mechanism of malic enzyme (Chang & Hsu. 1977a: Pry & Hsu. 1978). However. bromopyruvate modifies active site SH only by utilizing differential labeling technique. Without the protection of substrate analogue tartronate and other cofactors, bromopyruvate actually was non-specific for the active site or for the group modified (Chang & Hsu, 1977a). We demonstrated in the present study the satisfactory specificity of oxidized NADP for malic enzyme. This reagent could specifically form a labile Schiff base with an active site lysine. After being stabilized with NaBH,, lysine has been identified as the only amino acid being modified. The site specificity of oxidized NADP was demonstrated in the following experiments: (a) The inactivation by oxidized NADP showed saturation kinetics, suggesting the formation of an enzyme-reagent complex. The strict structural requirement for the inactivator was demonstrated by the finding that oxidized NAD. having four available reactive aldehyde groups. inactivated the enzyme at a rate only l/7 that of oxidized NADP under the same conditions; (b) Oxidized NADP could replace NADP as a coenzyme. with a K, value of 30pM. Oxidized NADP was also found to be a competitive inhibitor vs NADP with Ki value of 37 /tM. The excellent agreement between the K, and Ki values indicates that oxidized NADP is bound at the nucleotide site as suggested by its NADP-like structure; (c) Oxidized NADP inactivation was protected by nucleotides: (d) Extensively modified enzyme showed only 0.46 equivalent incorporation per enzyme site. The reagent incorporation was prevented by NADP. Therefore we conclude that oxidized NADP is a more specific affinity label than bromopyruvate. and would be a better instrument for the exploration on the geometry of the active centre of malic enzyme. Oxidized nucleotides have been used rather successfully as an affinity label for a number of
625
other enzymes (Bellini et al.. 1979; Fayat et al., 1978; Easterbrook-Smith et al., 1976; Ranieri-Raggi et al., 1976; Rippa et al.. 1974). We believe that oxidized nucleotides will be ideal affinity labels with both site and group specificities for all other enzymes using a nucleotide as their substrate or inhibitor. Equilibrium dialysis (Pry & Hsu, 1980) and chemical modification studies (Chang & Hsu. 1977) suggested the participation of histidine residue in NADPH binding. Kinetic studies (Schimerlik & Cleland. 1977) on the pH profile of V/K for NADP showed a dependence of NADP binding on the ionization of a group with PK of 5.3 and the protonation of a group with a pK of 9.3. However. attempts by Pry & Hsu (1980) to identify these groups by direct binding studies were unsuccessful. due to the instability of both malic enzyme and NADP at extreme pH value. The group with pK 5.3 may be assigned to histidine residue. Our present results indicate the involvement of lysine residue in the nucleotide binding. The group with pK 9.3 may thus be assigned to lysine. However, since oxidized NADP is a bulky group. the possibility that the impairment of nucleotide binding was the result of a sterlc effect could not be completely ruled out. The nucleotide binding was further studied by characterizing the inhibitory action of various NADP or NAD analogues on the malic enzyme-catalyzed NADP reduction. After examining the results shown in Table 2, two features were noticed: (a) only the NADP analogues with 2’-phosphate group in the ribose ring bound to the adenine (adenosine 2’S’-bisphosphate, oxidized NADP) inhibited the enzyme competitively with respect to NADP. The NAD analogues were non-competitive inhibitor for malic enzyme; (b) the competitive inhibitors had much lower inhibition constants. suggesting higher binding affinity. The inhibition constants for the non-competitive inhibitors were at least one order of magnitude higher. These results suggest that the 2’-phosphate group is important for the binding between NADP and malic enzyme. Removal of the 2’-phosphate group from the NADP molecule (NAD) drastically reduced its ability to function as a coenzyme for this enzyme. The NAD-catalyzed enzyme activity was only l.2”,, that of the NADP-catalyzed activity. How-
C NADPHI
(PM)
Fig. 6. Fluorimetric titration of the native and oxidizedNADP-modified malic enzyme with NADPH. All cuvettes contained 62 mM Tris- HCI buffer (pH 7.0) and the following: (0). without enzyme; (0). the native enzyme: (0). oxidized-NADP-modified enzyme with I?‘,, residual activity. The quenching due to reagents have been corrected.
Gu-GANG
626
ever, it is difficult to assign the involvement of lysine residue in the binding of 2’-phosphate or the pyrophosphate moiety from the protection experiment (Table 1). The exact role of lysine or histidine residues in the nucleotide binding will be the subject of further investigation. NADP has a K, value of 1.4pM (Hsu, Lardy & Cleland, 1967). Oxidation with sodium periodate resulted in the cleavage of the bond between carbon 2’ and 3’ of the ribose ring bound to nicotinamide and formation of two aldehyde groups at these carbons (Dallocchio et al., 1976). The much higher K, (30 PM) or Ki (37 PM) values for the oxidized NADP implies that oxidation resulted in decreased affinity between the nucleotide and the enzyme. This result could be due to the more flexible conformation of the nucleotide molecule after the ribose ring was opened. Recently, all kinetic or binding studies suggest the anti-cooperativity of pigeon liver malic enzyme. For example, EPR studies indicated the presence of two kinds of Mn’+ sites (Hsu et al., 1976). Mn2+ and Mg2+ also showed kinetic negative cooperativity at inhibitory levels of malate (Hsu er al., 1976; Hsu & Liu, 1980; Hsu & Pry, 1980; Schimerlik et al., 1977). The affinity label bromopyruvate alkylated two of the four “essential” SH groups (Chang & Hsu, 1977a). The unmodified SH groups reacted with non-specific reagents such as N-ethylmaleimide or 5,5’-dithiobis(2nitrobenzoic acid) more slowly than did the SH groups of the native enzyme (Pry & Hsu, 1978). Equilibrium binding studies showed that malate bound to two types of sites with differing affinity. The inhibitor oxalate bound to only two of the four available sites (Pry & Hsu, 1980). However, fluorimetric titration (Hsu & Lardy, 1967; Pry & Hsu, 1980) and equilibrium binding studies (Pry & Hsu, 1980) indicated that malic enzyme had four independent and equivalent nucleotide sites. Half-of-the-site reactivity for the nucleotide has been noted by fast kinetics (Reynolds et al., 1978). Our incorporation results represent another approach to the study of the nucleotide site. It should be noted that our determination depends upon knowing the specific activity of the reactive oxidized NADP. If only one-half of the labeled molecules can react with the enzyme, then the incorporation of reagent into the enzyme would be underestimated. However, since the reagent was routinely checked for purity by t.1.c. and no unoxidized nucleotide was found in the oxidized nucleotide preparations, we believe our conclusion probably to be correct. The half-site stoichiometry together with other results strongly suggest the negative cooperativity between subunits of malic enzyme and support the “half-ofthe-site” model proposed by Hsu & Pry (1980) to show that only two of the four identical, or perhaps unsymmetrical arranged identical subunits (Degani & Degani, 1980) of this enzyme undergo catalysis. SUMMARY
Pigeon liver malic enzyme was inactivated by periodate-oxidized NADP at neutral pH and room temperature, The inactivation showed saturation kinetics, suggesting the formation of an enzyme-reagent complex. Subsequent inactivation was due to the formation of a Schiff base between the aldehyde groups
CHANG et ul.
of the reagent and the e-amino group of the lysine residue, as identified by comparing with standards in thin layer chromatography. Complete protection of malic enzyme from oxidized NADP inactivation was afforded by NADP. Partial protection was also observed with other nucleotides. Oxidized NADP was also found to be a substrate (K, = 30nM) and a competitive inhibitor with respect to NADP (Ki = 37 PM) for this enzyme. The modified enzyme showed increased apparent Michaelis constants for NADP and NADPH but not for malate or pyruvate. The impairment of binding between the modified enzyme and NADPH was confirmed by fluorimetric titration. The incorporation of radioactivity from the oxidized-[3H]NADP gave a half-site stoichiometry, i.e., two molecules of reagent incorporated per enzyme tetramer, indicating a strong negative cooperativity between the four subunits of malic enzyme. The 2’-phosphate group seems to be essential for the binding between NADP and malic enzyme. Acknowledgements-We thank Professor Foo Pan for reading this manuscript. This work was supported in part by a grant (NSC-69B-0412-06-34) from the National Science Council, Republic of China.
REFERENCES
BELLING T., SIGNORINI M., DALLOCCHIO F. & RIPPA M. (1979) Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida &is. Biochem. J. 183, 297-302. CHANG G. G. & CHUEH S. H. (1980) Nonfunctional nature of sulfhydryl groups for pigeon liver malic enzyme. Int. J. Peptide Protein Res. 16, 321-326. CHANG G. G. & Hsu R. Y. (1973) The substrate analog bromopyruvate as a substrate, an inhibitor and an alkylating agent of malic enzyme of pigeon liver. Biochem. biophys. Res. Commun. 55, 58G587. CHANG G. G. & Hsu R. Y. (1977) Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride. Biochim. biophyb. Acta 483, 228-235. CHANG G. G. & Hsu R. Y. (1977a) Mechanism of pigeon liver malic enzyme. Kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate. Biochemistry 16, 31 l-320. CHANG G. G. & HUANG T. M. (1979) Oxidized NADP as a potential active-site-directed reagent of pigeon liver malic enzyme. Biochem. biophys. Rex Commun. 86, 829-836. CHANG G. G. & HUANG T. M. (1980) Involvement of tyrosyl residues in the substrate binding of pigeon liver malic enzyme. Biochim. biophys. Acta 611, 217-226. CHANG G. G. & HUANG T. M. (1981) Modification of essential arginine residues of pigeon liver malic enzyme. Biochim. biophys. Acta 660, 341-347. DALLOCCHIO F., NEGRINI R., SIGNORINI M. & RIPPA M. (1976) Identification of the chemical groups involved in the bindine. of neriodate-oxidized NADP’ to 6-phosphogluconate dehydrogenase. Biochim. biophys. Acta 429, 629-634. DEGANI Y. & DEC~ANIC. (1980) Enzymes with asymmetrically arranged subunits. Trends Biochem. Sci. 5, 337-341. DIXON M. & WEBB E. C. (1979) Enzymes 3rd edn p. 350. Academic Press, New York, U.S.A.
Pigeon liver malic enzyme EASTERBROOK-SMITH S. B., WALLACEJ. C. & KEECHD. B. (1976) Pyruvate carboxylase: Affinity labelling of the magnesium adenosine triphosphate binding site. Eur. J. Biochem. 62, 125130. FAYATG., FROMANTM. & BLANQUET S. (1978) AminoacyltRNA synthetase: Affinity labeling of the ATP binding site by 2’,3’-ribose oxidized ATP. Proc. narn Aced. Sci., U.S.A. 75, 2088-2092. Hsu R. Y. & LARDY H. A. (1967) Pigeon liver malic enzyme. II. Isolation, crystallization. and some properties. J. hiol. Chem. 242, 52@-526. Hsu R. Y. & LARDY H. A. (1967a) Pigeon liver malic enzyme. III. Fluorescence studies of &enzyme binding, J. ha)/. Them. 242, 5277532. Hsu R. Y.. LARDYH. A. & CLELANDW. W. (1967) Pigeon liver malic enzyme. V. Kinetic studies. J. biol. Chem. 242, 5315-5322. Hsu R. Y. & LIU N. (1980) Kinetic studies on the malic enzyme of pigeon liver: Inhibition of the Mg’+-activated oxidative decarboxytation reaction by L-matate. Fedn Proc. Fedn Am. Sots exp. Biol. 39, 1823. Hsu R. Y., MILDVANA. S., CHANG G. G. & FUNG C. H. (1976) Mechanism of pigeon liver malic enzyme. Magnetic resonance and kinetic studies of the role of Mn*+. J. biol. Chem. 251, 6574-6583. Hsu R. Y. & PRY T. A. (19801 Kinetic studies of the malic enzyme of pigeon liver. “Half-of-the-site” behavior of the the enzyme tetramer in catalysis and substrate inhibition. Biur~l~~~srr~ 19, 962--968. NEVALDINE B. H., BASSELA. R. & Hsv R. Y. (1974) Mechanism of pigeon liver matic enzyme. Subunit structure. Biochim. biophys. Arta 336, 283-293.
627
PRY T. A. & HSU R. Y. (1978) Mechanism of pigeon liver malic enzyme. Reactivity of class II sulthydryl groups as a conformational probe for the “half-of-the-site” reactivity of the enzyme with bromopyruvate. Biochemistry 17, 40244029.
PKY T. A. & HSU R. Y. (1980) Equilibrium substrate binding studies of the malic enzyme of pigeon liver. Equivalence of nucleotide sites and antico-operativity associated with the binding of t_-malate to the enzyme-manganese (II)-reduced nicotinamide adenine dinucleotide phosphate ternary complex. Biochemistry 19,951-962. RANIERI-RAGGIM. & RAGGI A. (1976) Specific moditication of the GTP binding sites of rat 5’-adenylic acid aminohydrolase by periodate-oxidized GTP. Biochim. biophys. Acfa 445, 223-233. REYNOLDSC. H., Hsu R. Y., MATTHEWB., PRY T. A. & DALZIEL K. (1978) Transient kinetic studies of malic enzyme. A conformational change associated with substrate inhibitor by malate. Archs Bioeh~m. B~op~~~s. 189, 309316. R~PPA M., SIGNORINIM., SIGNORIR. & DALLOCCH~O F. (1974) A new powerful inhibitor specific for the TPN binding site of 6-phosphogluconate dehydrogenase. FEBS Left. 51, 281-283. SCHIMERLIK M. I. & CLELANDW. W. (1977) nH variation of the kinetic parameters and the catalytic’ mechanism of malic enzyme. Biochemistry 16, 576-583. SCHIMERLIKM. I., GRIMSHAWC. E. & CLELANDW. W. (1977) Determination of the rate-Iimiting steps for malic enzyme by the use of isotope effects and other kinetic studies. Biocltemistrx 16, 571-575.
0020-711X/82/070621-07~3.00’0 Copyright 0 1982 Pergam~n Press Ltd
INVOLVEMENT OF LYSINE RESIDUE IN THE NUCLEOTIDE BINDING OF PIGEON LIVER MALIC ENZYME: MODIFICATION WITH AFFINITY LABEL PERIODATE-OXIDIZED NADP Department
Gu-GANG CHANG. TSU-CHUNG CHANT and TER-MEI HC’ANG of Biochemistry. National Defense Medical Center. Taipei. Taiwan. Republic (Rrceiced
16 Nocemher
of China
1981)
Abstract--l. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions. 2. The inactivation is due to the modification of an essential lysine residue. 3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification. 4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues. 5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased. 6. The binding between the modified enzyme and NADPH was impaired.
INTRODUCTION
sodium periodate. Sephadex G-10. NADP, NADPH. adcnosine Z’S’-bisphosphate, NAD. S-ADPribose, NMN. S-AMP, S-ADP, adenosine. adenine. nicotinamide, N-X-acetyl-lysine. o-glyceraldehyde (Sigma. U.S.A.); ethyleneglycol (Fischer. U.S.A.): silica gel (Camag U.S.A.);
Malic enzyme (t.-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). EC 1.1.1.40) from pigeon liver is a tetramer composed of probably identical subunits (Nevaldine er al.. 1974). Using chemical modification method, we have characterized the roles of cysteine (Chang & Hsu, 1977a; Chang & Chueh, 1980), histidine (Chang & Hsu, 1977), tyrosine (Chang & Huang, 1980), and arginine (Chang & Huang, 1981) residues in the mechanism of pigeon liver maiic enzyme. We have developed bromopyruvate as an affinity label for this enzyme (Chang & Hsu, 1973, 1977a). However, it was found that bromopyruvate actually modifies many non-essential groups outside the active centre as well. More recently. we introduced oxidized NADP as a potentially more specific aflkity label for this enzyme (Chang & Huang, 1979). In this communication, we wish to report the site and group specificities of this reagent. The results indicate that oxidized NADP is a more efficient affinity label than bromopyruvate for malic enzyme. Our results also suggest the involvement of lysine residue in the nucleotide binding of this enzyme. The half-of-the-site reactivity between nucleotide and malic enzyme is also presented.
MATERIALS
SF-5. Switzerland) were purchased from designated sources. All other chemicals were of reagent grade or better. Distilled, deionized water was used throughout this work. Pigeon liver maiic enzyme was purified according to Hsu & tardy (1967). The homogeneity of the purified enzyme was routinely checked by potyacrylamide gel efectrophoresis. The purified enzyme was dialyzed against 50 mM Tris-Cl buffer (pH 7.0 at 25’C) containing 107, glycerol and stored at -20°C. Protein concentration was determined at 278 nm. using an extinction coefficient of 0.86 for a O.ly,, (w/v) solution (Hsu & Lardy, 1967). M, 260.000 and 65.000 (Nevaldine er 01.. 1974) for the enzyme tetramer and monomer. respectively, were used for calculation of enzyme concentration.
The overall oxidative decarboxylase activity was assayed at 3o’C according to Hsu & Lardy (1967). The formation of NADPH was monitored continuously at 340nm. In some kinetic experiments. when nucleotide was used as the variable substrate, the assay was performed with a Gilford 250 spectrophotometer equipped with a IO-cm cuvette compartment. Cylindrical cuvettes with a total volume of 30 ml and IO-cm light path were used. Prepu~u&ion
AND METHODS
Mofrrials
C3H]Sodihm borohydride (Radiochemical Center England); t_-malic acid. sodium pyruvate (Calbiochem, Address for sending correspondence: Gu-gang Chang, Ph.D. Department of Biochemistry, National Defense Medical Center. P.O. Box 8244. Taipei, Taiwan, Republic of China. &C. 14 l--F
t$oxidked
NADP
Oxidation of NADP and NAD bv NafO, were nerformed essentially according to Easterbrook-Smith et’ a(.. 1976). The purity of the oxidized nucleotides was routinely checked by.t.l.cVas previously described (Chang & Huang. 1979). Radioactive oxidized NADP (6.64 mM. 5490 cpminmol) was prepared according to Bellini ef al. (1979). NADP was first treated with E3H]NaBH, (9.5 Ci/mmol) and then with periodate. The oxidized-[‘H]NADP was purified by Sephadex G-10 chromatography. Separation of oxidized621
Gu-GANG
622
[%]NADP and C3H]NaBH, was confirms by the observation of two radioactive peaks, The first half of the leading peak was pooled. The radioactivity was measured in a Packard 3320 liquid scintillation spectrometer. The purity was checked by t.1.c. (Chang & Huang, 1979). No un-oxidized nucleotide was detectable in the oxidized nucleotide preparations. Modification
ofmalic enzyme with oxidized NADP
Kinetic studies of the modification were carried out by adding freshly prepared oxidized NADP to enzyme solution in borate buffer at pH 7.5 and 24°C. Immediately after the addition of the reagent, small aliquot was withdrawn and assayed for the zero-time enzyme activity. At intervals, samples of the reaction mixture were removed and assayed for the residual enzyme activity. Further reaction of the enzyme with oxidized NADP was negligible after dilution with assay mixture containing substrate protectors. The enzyme incubated with borate buffer alone did not show any inactivation during the experimental period. The slope of the residual activity vs time semilog plot represents the observed pseudo-first order rate constant (k,,,). Protrcfion
sfudies
Protection experiments were performed as above except that the enzyme was preincubated with substrates or inhibitors for 10 min before adding oxidized NADP. The pH of substrate of inhibitor solution was adjusted to 7.5 before mixing with enzyme. The kohs of the inactivation was obtained from each experimental set. The percentage protection was catculated according to the equation:
- k,,,(protected)]/k,,,(unprotected); Identification NADP
x 100
of the amino ucid residue modified by oxidized
Malic enzyme was reacted with oxidized NADP as described above. The inactivated enzyme was reduced with a few crystals of C3H]NaBH,, dialyzed exhaustively against water, and then hydrolyzed with 6 N HCl in an evacuated. sealed ampoule at 110°C for 24 hr. The hydrolysate was subjected to t.1.c. on glass plate coated with silica gel in a solvent system of I-propanol-25% NH, (67:33, v/v) or I-butanol-acetic acid-water (60:20:20, v/v). After chromatography, the plate was dried, marks were made every 0.5 or 1 cm, and the gel of each section was removed and counted for radioactivity. The standards for the identification of the amino acid being modified were prepared from N-z-acetyl-lysine and ~-giycera~dehyde according to Dallocchio et al. (1976). Covalent
~ube~~~g qfmaiic enzyme with oxidized-[3H]NADP
CHANG er al.
was measured (Hsu & Lardy, 1967a). In the control, reagents except the enzyme were added for correction the quenching due to reagents,
all of
RESULTS malic enzyme NADP as a substrate or an inhibitor
Kinetic
properties
of
using oxidized
Oxidized NADP was previously shown to be reduced by malic enzyme (Chang & Huang, 1979). The reaction was linear with time and enzyme concentration. The apparent Michaelis constant for oxidized NADP (30 ) 2 PM) was higher than that for NADP (1 c 3 PM), with only l/10 V,,,,, of the regular assay. Oxidized NADP was shown as a non-competitive inhibitor vs t.-malate in oxidative decarboxylation. This compound also inhibited NADP reduction competitively with a ki value of 3711M (Fig. 1). indicating binding at the nucleotide site.
ln~~~i~~t~on o~malic enzyme by oxidized NADP Incubation of malic enzyme with oxidized NADP at neutral pH and 24°C resulted in a rapid loss of enzyme activity. The inactivation reaction exhibited pseudo-first order kinetics until 907,; inactivation, but the kinetics were not consistent with any simple scheme after longer-time incubation. The enzyme always retained some residual enzymatic activity after prolonged incubation. The dependence of the loss of enzyme activity on oxidized NADP concentration has been studied (Chang & Huang, 1979). A plot of kobs vs oxidized NADP concentration showed saturation behavior, and the double reciprocal plot was linear. The results suggested the formation of an enzyme-oxidized NADP binary complex before inactivation process, and oxidized NADP acts as an affinity label for malic enzyme. The structural requirement of oxidized NADP as an effective inactivator for malic enzyme was demonstrated in Fig. 2. Oxidized NAD, under the same conditions, inactivated the enzyme much slower. The kobs for oxidized NADP and oxidized NAD were 0.06 and 0.0087 min- ‘, respectively. We found that the NADcatalyzed enzyme activity was only 1.20,; that of the regular assay. However, NAD was found to be an
7
Malic enzyme (0.18 mg) was incubated with freshly prepared oxidized-[3H]NADP (0.05 _ 0.5 mM, 1.8 z 18 x 10s cpm) in 15 mM borate buffer (pH 7.5) at 24°C. Aliquot samples were removed at zero-time and after 1 hr incubation. A few crystals of NaBH, was added to stop the reaction and reduce the covalent bond. The samples were dialyzed against water to removed unbound radioactivity, and analyzed for protein-bound radioactivity. The incorporation of covalent labei was expressed as oxidized-[3HfNADP per enzyme subunit. In some experiments, malic enzyme was preincubated with NADP to protect the nucleotide site. The protected enzyme was then treated with oxidized-[sH]NADP as described above. I / C NADP
Fluorescence
1
t I.‘MI
titration
Fluorescence titration of the native or oxidized-NADPinactivated enzyme with NADPH was performed using an Aminco-Bawman spectrofluorimeter. The nucleotidc was excited at 350 nm and the emission fiuoreseence at 4.50 nm
Fig. 1. Inhibition of mahc enzyme by oxidized NADP. The enzyme activities were assayed at different levels of oxidized NADP as described in Materials and Methods with NADP as the variable substrate.
Pigeon
liver malic enzyme
623
Table 2. Inhibition
Inhibition
Analogue
0
IO
20 TIME
30
40
(man)
Fig. 2. Inactivation of malic enzyme by oxidized NADP and oxidized NAD. Malic enzyme (63pg) was incubated with 0.98 mM oxidized NADP (0) or oxidized NAD (a) in 40 mM borate buffer (pH 7.5) at 24’C. The logarithm of residual activity was plotted against time.
effective non-competitive (see next section).
inhibitor
for this
enzyme
Protection of the enzyme from inactivation by oxidized NADP Table 1 summarizes the effects of nucleotides and Full protection their analogs on inactivation. (99 f 2% in six experiments) was afforded by 8.8 mM NADP. Other nucleotides or divalent metal ions gave partial protection. Substrates malate or pyruvate alone did not give any protection, but enhanced the protective effect of lower concentration of NADP. The protection by nucleotide coenzyme strongly suggests that modification was at the nucleotide binding site. Further protective experiments were carried out to determine which portion of the NADP molecule was responsible for the protection. NAD or its analogs AMP, ADP, NMN, adenosine, nicotinamide and adenine were all found to be non-competitive inhibitors of malic enzyme (Table 2), whereas the NADP analogs adenosine 2’,5’-bisphosphate or oxidized NADP were competitive inhibitor with respect to NADP, Table 1. Effects of nucleotide the
rate
of inactivation
Addition NADP
NADPH Adenosine 2’,5’-bisphosphate NAD 5’-ADP 5’-AMP NMN Adenosine
coenzymes and inhibitors on of malic enzyme by oxidized NADP
Concentration (mM)
y0 Protection
8.8 7.0 4.5 3.7 0.9 3.7
99 92 89 79 46 86
7.0 0.9 8.8 8.8 8.8 8.8 8.8
58 46 93 90 68 79 71
Conditions were as described in Fig. 2, except that the enzyme was preincubated with the various additions listed.
of pigeon liver malic enzyme by NADP or NAD analogues
Adenosine 2’,5’-bisphosphate Oxidized NADP 5’-ADP-ribose NAD 5’-AMP 5’-ADP NMN Adenosine Adenine Nicotinamide
pattern*
Competitive Competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive Non-competitive
Kit (mM)
0.006 0.037: 0.6 1.1 3.8 4.2 7.1 10 33 231
* Double reciprocal plot was used to examine the inhibition pattern. t Dixon plot (Dixon & Webb, 1979) was used to evaluate the inhibition constant. $ From Fig. I. much lower Ki value than that for the NAD analogs. In examination of the structure of the inhibitors that gave protection (NADP, NADPH, adenosine 2’,5’-bisphosphate, NAD. AMP, ADP, NMN and adenosine), with the only exception of adenosine, all compounds contain a phosphoryl moiety. Nicotinamide, adenine, Pi and PP, had no effect on the rate of inactivation (data not shown). These results suggest that phosphoryl binding site was attacked by oxidized NADP. The lack of any protective effect of Pi and PP, was probably due to its inability to bind to the enzyme. The protective effect of adenosine could be due to a steric effect. and have
Identification
of the modijied amino acid
The inactivation of malic enzyme by oxidized NADP was reversible by dilution and could be made irreversible by treating the modified enzyme with NaBH, (Chang & Huang, 1979). These results strongly suggested the formation of a Schiff base between the aldehyde groups of oxidized NADP and lysyl e-amino group of the enzyme. The reversibility suggested that the inactivation was not due to a gross structural change, although local conformational change could not be ruled out. The exact nature of the amino acid being modified was identified as lysine as anticipated (Fig. 3). A modified enzyme sample was reduced with [“H]NaBH, to label the modified amino acid residue. Figure 3B shows the t.1.c. chromatogram of the acid hydrolysate developed in I-propanol: 25% NH,. The modified enzyme gave one major and one minor radioactive peaks with R, values of 0.69 and 0.84, respectively. Figure 3A shows the chromatogram of standard prepared for identification. The complete match of these two chromatograms indicates specific labeling of lysine residue by this method. However, compared to the reported values (Bellini et al., 1979; Dallocchio er al., 1976), the R, values were slightly higher in our samples probably due to different quality of the solvents. Similar results were obtained with the 1-butanol-acetic acidwater solvent system. Incubation of a SH-masked enzyme with oxidized NADP also caused inactivation (data not shown), indicating that the inactivation was
C&J-GANG CHANG C[ al
3#-OXlDIZED
MIGRATION
(cm1
Fig. 3. Thin layer chromatogram of the acid hydrolysate of oxidized-NADP-modified enzyme. Malic enzyme (0.36 mg) was incubated with 1.2mM oxidized NADP in 22mM borate buffer (pH 7.5) at 24 C for 1 hr. The inactivated enzyme (residual activity, 7”,,1 was reduced with [‘HJNaBH,, dialyzed and hydrolyzed. The standard (AI and hydrolysate (B) were chromatographied as described in Materials and Methods.
not clue to SH modification. So far lysine was the only amino acid found to be modified with oxidized nucleotides in some other enzymes (Bellini et nl., 1979; Dallocchio et al.. 1976: Easterbrook-Smith et al.. 1976). The attempts to prepare a reduced Schick base between oxidized nucleotide and arginine (Bellini rt ul.. 1979: Easterbrook-Smith et al., 1976). cysteine or histidine (Easterbrook-Smith et ul.. 1976) were not successful.
Stoichiometry of the labeling The stoichiometry was studied by incubating the malic enzyme with oxidized-[3H]NADP and reduced with NaBH,. Figure 4 shows the incorporation of oxidized-C3H]NADP to malic enzyme. A linear plot of residual activity vs [3H]incorporation gave 0.46 molecule of oxidized NADP per enzyme subunit upon extrapolation to complete inactivation. Prolonged incubation did not give further incorporation (indicated by an arrow in Fig. 4). NADP could pre-
Fig. 4. Correlation of inactivatlon and radioactivity incorporation. Malic enzyme was incubated with ouidized-[3H]NADP as described in Materials and Methods in the presence (a) or absence (0) of NADP. Enzyme activity and radioactivity incorporation were determined and calculated. The result obtained from a prolonged incubation (5 hr) was indicated by an arrow,
vent the [3H]incorporation, as well as protect the enzyme from inactivation. This result directly demonstrated the site specificity of oxidized NADP.
Figure 5 shows the effects of oxidized NADP modification on the kinetic parameters of nucleotide coenzymes. The apparent I<,,, for NADP was increased 5 _ &fold (from 1.2 + 0.3 FM to 6.4 + 0.4 FM) and that for NADPH increased Z.&fold (from 2 PM to 5.6pM) after modification. The K,,, Lalues for other substrates (malate, pyruvate) were not changed after modification (Table 3). The V,_ of the enzyme after modification was decreased (Fig. 5). These results suggest that a decrease in binding afFinity between the modified enzyme and nucleotide coenzyme was probably the cause of decreased enzyme activity. Direct evidence for the involvement of lysine residue in the nucleotide binding was provided by fluorescence titration experiments as shown in Fig. 6. Titration of the native enzyme with NADPH resulted in a binary complex formation and an enhancement of nuclcotide fluorescence.
Oxidized-NADP-modified
0 01 I /
Fig. 5. Kinetic
properties
with
0,203
CNADPHI
enzyme
04 05 ((IM-‘)
of the native and oxidized-NADP-modified enzyme. The native enzyme (2.8 pg enzyme (5.6 pg with 24”” residual activity in A and IO”:, residual activity in 8) (0) were assayed with nucleotide as the variable substrate. Velocities were calculated for the same amount of enzyme.
in A. 3.5 pg in B) (0). or oxidized-NADP-modified
8.7pg
NAOP INCORPORATED/%WNIT
failed
Pigeon Table
3. Apparent Michaelis NADP-modified Native
Substrate t-malate UADP Pyruvate YADPtI
liver malic enzyme
constants of the oxidizedmalic enzyme
enzyme
Modified
Specific* activity
K,t (PM)
Specific* activity
44 41 46 45 1.9 1.5
67 56 1.5 0.9 20 X 103 2
1.3 1.6 0.9 II 1.2 0.15
enzyme
67 66 1.3 5.5 21 x IO3 5.6
* Overall oxidative decarboxylase was assayed when t.-malatr or NADP was the variable substrate. Reductase partlnl activity was assayed when pyruvate or NADPH was the variable substrate. t Double reciprocal plot was used to evaluate the Michaelis constant.
to enhance the NADPH fluorescence, lack of complex formation.
indicating
the
DISCUSSION
In previous studies. bromopyruvate was shown to be a useful affinity label in the study of reaction mechanism of malic enzyme (Chang & Hsu. 1977a: Pry & Hsu. 1978). However. bromopyruvate modifies active site SH only by utilizing differential labeling technique. Without the protection of substrate analogue tartronate and other cofactors, bromopyruvate actually was non-specific for the active site or for the group modified (Chang & Hsu, 1977a). We demonstrated in the present study the satisfactory specificity of oxidized NADP for malic enzyme. This reagent could specifically form a labile Schiff base with an active site lysine. After being stabilized with NaBH,, lysine has been identified as the only amino acid being modified. The site specificity of oxidized NADP was demonstrated in the following experiments: (a) The inactivation by oxidized NADP showed saturation kinetics, suggesting the formation of an enzyme-reagent complex. The strict structural requirement for the inactivator was demonstrated by the finding that oxidized NAD. having four available reactive aldehyde groups. inactivated the enzyme at a rate only l/7 that of oxidized NADP under the same conditions; (b) Oxidized NADP could replace NADP as a coenzyme. with a K, value of 30pM. Oxidized NADP was also found to be a competitive inhibitor vs NADP with Ki value of 37 /tM. The excellent agreement between the K, and Ki values indicates that oxidized NADP is bound at the nucleotide site as suggested by its NADP-like structure; (c) Oxidized NADP inactivation was protected by nucleotides: (d) Extensively modified enzyme showed only 0.46 equivalent incorporation per enzyme site. The reagent incorporation was prevented by NADP. Therefore we conclude that oxidized NADP is a more specific affinity label than bromopyruvate. and would be a better instrument for the exploration on the geometry of the active centre of malic enzyme. Oxidized nucleotides have been used rather successfully as an affinity label for a number of
625
other enzymes (Bellini et al.. 1979; Fayat et al., 1978; Easterbrook-Smith et al., 1976; Ranieri-Raggi et al., 1976; Rippa et al.. 1974). We believe that oxidized nucleotides will be ideal affinity labels with both site and group specificities for all other enzymes using a nucleotide as their substrate or inhibitor. Equilibrium dialysis (Pry & Hsu, 1980) and chemical modification studies (Chang & Hsu. 1977) suggested the participation of histidine residue in NADPH binding. Kinetic studies (Schimerlik & Cleland. 1977) on the pH profile of V/K for NADP showed a dependence of NADP binding on the ionization of a group with PK of 5.3 and the protonation of a group with a pK of 9.3. However. attempts by Pry & Hsu (1980) to identify these groups by direct binding studies were unsuccessful. due to the instability of both malic enzyme and NADP at extreme pH value. The group with pK 5.3 may be assigned to histidine residue. Our present results indicate the involvement of lysine residue in the nucleotide binding. The group with pK 9.3 may thus be assigned to lysine. However, since oxidized NADP is a bulky group. the possibility that the impairment of nucleotide binding was the result of a sterlc effect could not be completely ruled out. The nucleotide binding was further studied by characterizing the inhibitory action of various NADP or NAD analogues on the malic enzyme-catalyzed NADP reduction. After examining the results shown in Table 2, two features were noticed: (a) only the NADP analogues with 2’-phosphate group in the ribose ring bound to the adenine (adenosine 2’S’-bisphosphate, oxidized NADP) inhibited the enzyme competitively with respect to NADP. The NAD analogues were non-competitive inhibitor for malic enzyme; (b) the competitive inhibitors had much lower inhibition constants. suggesting higher binding affinity. The inhibition constants for the non-competitive inhibitors were at least one order of magnitude higher. These results suggest that the 2’-phosphate group is important for the binding between NADP and malic enzyme. Removal of the 2’-phosphate group from the NADP molecule (NAD) drastically reduced its ability to function as a coenzyme for this enzyme. The NAD-catalyzed enzyme activity was only l.2”,, that of the NADP-catalyzed activity. How-
C NADPHI
(PM)
Fig. 6. Fluorimetric titration of the native and oxidizedNADP-modified malic enzyme with NADPH. All cuvettes contained 62 mM Tris- HCI buffer (pH 7.0) and the following: (0). without enzyme; (0). the native enzyme: (0). oxidized-NADP-modified enzyme with I?‘,, residual activity. The quenching due to reagents have been corrected.
Gu-GANG
626
ever, it is difficult to assign the involvement of lysine residue in the binding of 2’-phosphate or the pyrophosphate moiety from the protection experiment (Table 1). The exact role of lysine or histidine residues in the nucleotide binding will be the subject of further investigation. NADP has a K, value of 1.4pM (Hsu, Lardy & Cleland, 1967). Oxidation with sodium periodate resulted in the cleavage of the bond between carbon 2’ and 3’ of the ribose ring bound to nicotinamide and formation of two aldehyde groups at these carbons (Dallocchio et al., 1976). The much higher K, (30 PM) or Ki (37 PM) values for the oxidized NADP implies that oxidation resulted in decreased affinity between the nucleotide and the enzyme. This result could be due to the more flexible conformation of the nucleotide molecule after the ribose ring was opened. Recently, all kinetic or binding studies suggest the anti-cooperativity of pigeon liver malic enzyme. For example, EPR studies indicated the presence of two kinds of Mn’+ sites (Hsu et al., 1976). Mn2+ and Mg2+ also showed kinetic negative cooperativity at inhibitory levels of malate (Hsu er al., 1976; Hsu & Liu, 1980; Hsu & Pry, 1980; Schimerlik et al., 1977). The affinity label bromopyruvate alkylated two of the four “essential” SH groups (Chang & Hsu, 1977a). The unmodified SH groups reacted with non-specific reagents such as N-ethylmaleimide or 5,5’-dithiobis(2nitrobenzoic acid) more slowly than did the SH groups of the native enzyme (Pry & Hsu, 1978). Equilibrium binding studies showed that malate bound to two types of sites with differing affinity. The inhibitor oxalate bound to only two of the four available sites (Pry & Hsu, 1980). However, fluorimetric titration (Hsu & Lardy, 1967; Pry & Hsu, 1980) and equilibrium binding studies (Pry & Hsu, 1980) indicated that malic enzyme had four independent and equivalent nucleotide sites. Half-of-the-site reactivity for the nucleotide has been noted by fast kinetics (Reynolds et al., 1978). Our incorporation results represent another approach to the study of the nucleotide site. It should be noted that our determination depends upon knowing the specific activity of the reactive oxidized NADP. If only one-half of the labeled molecules can react with the enzyme, then the incorporation of reagent into the enzyme would be underestimated. However, since the reagent was routinely checked for purity by t.1.c. and no unoxidized nucleotide was found in the oxidized nucleotide preparations, we believe our conclusion probably to be correct. The half-site stoichiometry together with other results strongly suggest the negative cooperativity between subunits of malic enzyme and support the “half-ofthe-site” model proposed by Hsu & Pry (1980) to show that only two of the four identical, or perhaps unsymmetrical arranged identical subunits (Degani & Degani, 1980) of this enzyme undergo catalysis. SUMMARY
Pigeon liver malic enzyme was inactivated by periodate-oxidized NADP at neutral pH and room temperature, The inactivation showed saturation kinetics, suggesting the formation of an enzyme-reagent complex. Subsequent inactivation was due to the formation of a Schiff base between the aldehyde groups
CHANG et ul.
of the reagent and the e-amino group of the lysine residue, as identified by comparing with standards in thin layer chromatography. Complete protection of malic enzyme from oxidized NADP inactivation was afforded by NADP. Partial protection was also observed with other nucleotides. Oxidized NADP was also found to be a substrate (K, = 30nM) and a competitive inhibitor with respect to NADP (Ki = 37 PM) for this enzyme. The modified enzyme showed increased apparent Michaelis constants for NADP and NADPH but not for malate or pyruvate. The impairment of binding between the modified enzyme and NADPH was confirmed by fluorimetric titration. The incorporation of radioactivity from the oxidized-[3H]NADP gave a half-site stoichiometry, i.e., two molecules of reagent incorporated per enzyme tetramer, indicating a strong negative cooperativity between the four subunits of malic enzyme. The 2’-phosphate group seems to be essential for the binding between NADP and malic enzyme. Acknowledgements-We thank Professor Foo Pan for reading this manuscript. This work was supported in part by a grant (NSC-69B-0412-06-34) from the National Science Council, Republic of China.
REFERENCES
BELLING T., SIGNORINI M., DALLOCCHIO F. & RIPPA M. (1979) Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida &is. Biochem. J. 183, 297-302. CHANG G. G. & CHUEH S. H. (1980) Nonfunctional nature of sulfhydryl groups for pigeon liver malic enzyme. Int. J. Peptide Protein Res. 16, 321-326. CHANG G. G. & Hsu R. Y. (1973) The substrate analog bromopyruvate as a substrate, an inhibitor and an alkylating agent of malic enzyme of pigeon liver. Biochem. biophys. Res. Commun. 55, 58G587. CHANG G. G. & Hsu R. Y. (1977) Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride. Biochim. biophyb. Acta 483, 228-235. CHANG G. G. & Hsu R. Y. (1977a) Mechanism of pigeon liver malic enzyme. Kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate. Biochemistry 16, 31 l-320. CHANG G. G. & HUANG T. M. (1979) Oxidized NADP as a potential active-site-directed reagent of pigeon liver malic enzyme. Biochem. biophys. Rex Commun. 86, 829-836. CHANG G. G. & HUANG T. M. (1980) Involvement of tyrosyl residues in the substrate binding of pigeon liver malic enzyme. Biochim. biophys. Acta 611, 217-226. CHANG G. G. & HUANG T. M. (1981) Modification of essential arginine residues of pigeon liver malic enzyme. Biochim. biophys. Acta 660, 341-347. DALLOCCHIO F., NEGRINI R., SIGNORINI M. & RIPPA M. (1976) Identification of the chemical groups involved in the bindine. of neriodate-oxidized NADP’ to 6-phosphogluconate dehydrogenase. Biochim. biophys. Acta 429, 629-634. DEGANI Y. & DEC~ANIC. (1980) Enzymes with asymmetrically arranged subunits. Trends Biochem. Sci. 5, 337-341. DIXON M. & WEBB E. C. (1979) Enzymes 3rd edn p. 350. Academic Press, New York, U.S.A.
Pigeon liver malic enzyme EASTERBROOK-SMITH S. B., WALLACEJ. C. & KEECHD. B. (1976) Pyruvate carboxylase: Affinity labelling of the magnesium adenosine triphosphate binding site. Eur. J. Biochem. 62, 125130. FAYATG., FROMANTM. & BLANQUET S. (1978) AminoacyltRNA synthetase: Affinity labeling of the ATP binding site by 2’,3’-ribose oxidized ATP. Proc. narn Aced. Sci., U.S.A. 75, 2088-2092. Hsu R. Y. & LARDY H. A. (1967) Pigeon liver malic enzyme. II. Isolation, crystallization. and some properties. J. hiol. Chem. 242, 52@-526. Hsu R. Y. & LARDY H. A. (1967a) Pigeon liver malic enzyme. III. Fluorescence studies of &enzyme binding, J. ha)/. Them. 242, 5277532. Hsu R. Y.. LARDYH. A. & CLELANDW. W. (1967) Pigeon liver malic enzyme. V. Kinetic studies. J. biol. Chem. 242, 5315-5322. Hsu R. Y. & LIU N. (1980) Kinetic studies on the malic enzyme of pigeon liver: Inhibition of the Mg’+-activated oxidative decarboxytation reaction by L-matate. Fedn Proc. Fedn Am. Sots exp. Biol. 39, 1823. Hsu R. Y., MILDVANA. S., CHANG G. G. & FUNG C. H. (1976) Mechanism of pigeon liver malic enzyme. Magnetic resonance and kinetic studies of the role of Mn*+. J. biol. Chem. 251, 6574-6583. Hsu R. Y. & PRY T. A. (19801 Kinetic studies of the malic enzyme of pigeon liver. “Half-of-the-site” behavior of the the enzyme tetramer in catalysis and substrate inhibition. Biur~l~~~srr~ 19, 962--968. NEVALDINE B. H., BASSELA. R. & Hsv R. Y. (1974) Mechanism of pigeon liver matic enzyme. Subunit structure. Biochim. biophys. Arta 336, 283-293.
627
PRY T. A. & HSU R. Y. (1978) Mechanism of pigeon liver malic enzyme. Reactivity of class II sulthydryl groups as a conformational probe for the “half-of-the-site” reactivity of the enzyme with bromopyruvate. Biochemistry 17, 40244029.
PKY T. A. & HSU R. Y. (1980) Equilibrium substrate binding studies of the malic enzyme of pigeon liver. Equivalence of nucleotide sites and antico-operativity associated with the binding of t_-malate to the enzyme-manganese (II)-reduced nicotinamide adenine dinucleotide phosphate ternary complex. Biochemistry 19,951-962. RANIERI-RAGGIM. & RAGGI A. (1976) Specific moditication of the GTP binding sites of rat 5’-adenylic acid aminohydrolase by periodate-oxidized GTP. Biochim. biophys. Acfa 445, 223-233. REYNOLDSC. H., Hsu R. Y., MATTHEWB., PRY T. A. & DALZIEL K. (1978) Transient kinetic studies of malic enzyme. A conformational change associated with substrate inhibitor by malate. Archs Bioeh~m. B~op~~~s. 189, 309316. R~PPA M., SIGNORINIM., SIGNORIR. & DALLOCCH~O F. (1974) A new powerful inhibitor specific for the TPN binding site of 6-phosphogluconate dehydrogenase. FEBS Left. 51, 281-283. SCHIMERLIK M. I. & CLELANDW. W. (1977) nH variation of the kinetic parameters and the catalytic’ mechanism of malic enzyme. Biochemistry 16, 576-583. SCHIMERLIKM. I., GRIMSHAWC. E. & CLELANDW. W. (1977) Determination of the rate-Iimiting steps for malic enzyme by the use of isotope effects and other kinetic studies. Biocltemistrx 16, 571-575.