Photosynthesis Research 28: 69-76, 1991. © 1991 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Analogues of N A D P + as inhibitors and coenzymes for N A D P + malic e n z y m e from maize leaves Claudia P. Spampinato 1, Piotr Paneth 2., Marion H. O'Leary 2 & Carlos S. Andreo 1.

1Centro de Estudios Fotosint~ticos y Bioquimicos, UNR-CONICET, Suipacha 531, 2000 Rosario, Argentina; 2Department of Biochemistry, University of Nebraska-Lincoln, East Campus, Lincoln, Nebraska, 68583-0718, USA Received 29 December 1990; accepted 1 April 1991

Key words:

C4-photosynthesis, decarboxylation, NADP+-ME type

Abstract

Structural analogues of the NADP ÷ were studied as potential coenzymes and inhibitors for NADP ÷ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N6-etheno-nicotinamide adenine dinucleotide phosphate ( E N A D P + ) , 3-acetylpyridine-adenine dinucleotide phosphate (APADP + ), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP ÷) and/3-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate (2'3'NADPc ÷) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vm,x for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP ÷ ), 3-aminopyridine-adenine dinucleotide phosphate (AADP ÷ ), adenosine 2'-monophosphate (2'AMP) and adenosine 2': 3'-cyclic monophosphate (2'3'AMPc) were competitive inhibitors with respect to NADP ÷, while /3-nicotinamide adenine dinucleotide 3'-phosphate (3'NADP+), N A D ÷, adenosine 3'-monophosphate (3'AMP), adenosine 2':5'-cyclic monophosphate (2'5'AMPc), 5'AMP, 5 ' A D P , 5'ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical s e l f - consistent f i e l d - molecular orbitals calculations, suggest that the 2'-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C a atom of the pyridine ring is the major factor that governs the coenzyme activity.

Abbreviations:

E NADP ÷ -1,N6-etheno-nicotinamide adenine dinucleotide phosphate; N H D P ÷ nicotinamide-hypoxanthine dinucleotide phosphate; APADP ÷ - 3-acetylpyridine-adenine dinucleotide phosphate; SNADP ÷ -thionicotinamide-adenine dinucleotide phosphate; A A D P ÷ -3-aminopyridineadenine dinucleotide phosphate; 2'3'NADPc ÷-/3-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate; 3'NADP ÷-/3-nicotinamide adenine dinucleotide 3'-phosphate; 2 ' A M P - a d e n o s i n e 2'-monophosphate; 3'AMP - adenosine 3'-monophosphate; 2'3'AMPc - adenosine 2': 3' monophosphate cyclic; A - a d e n o s i n e ; R u B P - r i b u l o s e 1,5-bisphosphate; SCF-MO-Self-Consistent FieldMolecular Orbitals (method)

*On leave from the Institute of Applied Radiation Chemistry, Technical University of Lodz, Poland. *To whom correspondence should be addressed. Recipient of a Fellowshipfrom the John Simon Guggenheim Memorial Foundation.

Introduction

NADP+-malic enzyme [L-malate: NADP + oxidoreductase (decarboxylating); EC: 1.1.1.40]

70 catalyses the oxidative decarboxylation of malate in the presence of a divalent metal ion (presumably Mg 2+ in vivo) Malate + NADP + .~ Pyruvate

+ CO 2

+ NADPH This enzyme occurs in almost all living organisms including animals (Frenkel 1975) and higher plants (Asami et al. 1979), although its metabolic functions are different depending on the organism (Frenkel 1975, Asami et al. 1979). The malic enzyme from pigeon liver has been extensively studied (Hsu 1982), and the mechanism of reaction, role of metal ions, aminoacid residues at the active site and the interaction of NADP ÷ analogues with the enzyme have been well documented (Hsu 1982, Hsu et al. 1976, Vernon and Hsu 1983, Lee and Chang 1987, Hsu and Pry 1980, Schimerlik et al. 1977). NADP+-malic enzyme is found in many tissues of C 3, C 4 and CAM plants. The enzyme has been purified and characterized from various plant tissues (Asami et al. 1979, Iglesias and Andreo 1989, Iglesias and Andreo 1990a,b, Drincovich et al. 1990). Studies using fluorescence quenching suggest a rather complex mode of the binding of NADP ÷ to the enzyme, and this binding is influenced by the presence of either Mg 2+ or L-malate (Andreo et al. 1990). The concentration of NADP+-malic enzyme in the leaves of some C 4 plants is about 50-fold higher than that in C 3 plants (Slack and Hatch 1967). This enzyme plays a key role in the CO 2 concentrating mechanism in some C a plants (Edwards and Huber 1981). This is the primary decarboxylating enzyme responsible for releasing carbon dioxide in bundle sheath chloroplasts where it is eventually fixed by RuBP carboxylase. Chemical modification studies of the pure enzyme using diethylpyrocarbonate (Asami et al. 1979, Iglesias and Andreo 1989) reveal the presence of essential residues for catalytic functions (Jawali and Bhagwat 1987). However, the reaction mechanism of the enzyme is not fully understood. In the present report, kinetic techniques were used to investigate the pyridine nucleotide specificity of maize malic enzyme. The results

indicate that the 2' phosphate group was important for NADP + binding whereas the carboxamide carbonyl group of the nicotinamide moiety is necessary for coenzyme activity. Semiempirical calculations were performed with the hope that some of geometrical parameters might be correlated with the coenzyme activity. While no geometrical correlation was found, calculations indicated that the charge distribution within the nicotinamide moiety governs coenzyme activity. The positive charge on carbon C 4 of the pyridine ring seems to be essential for this activity.

Materials and methods

Chemicals. NADP +, E NADP +, NHDP +, APADP+, SNADP+, AADP+, 3'NADP+, N A D +, 2'3'NADPc +, 2'AMP, 3'AMP, 5'AMP, 2'5'AMPc, 2'3'AMPc, 5'ADP, 5'ATP and A were purchased from Sigma Chemical Co. Other chemicals were all of reagent grade. Plant material. Plants of Zea mays L. were grown outdoors (approximately 14h light daily; temperature 25-35 °C and 15-25 °C night). Mature leaves from plants about five weeks old were used. Enzyme purification and assay. NADP+-malic enzyme from maize leaves was purified by a procedure previously described (Iglesias and Andreo 1989). Enzyme activity was determined spectrophotometrically at 30 °C by monitoring N A D P H production at 340 nm in a Hitachi 150-20 spectrophotometer. The standard assay medium contained Tricine (50 mM) buffer pH 8, 4 mM Lmalate, 10mMMgC12 , 0.5 mM NADP + and malic enzyme in a final volume of 1 ml. One unit of enzyme is defined as the amount that catalyses the formation of 1/xmol of N A D P H per minute under the specified conditions. Initial-velocity studies were performed by varying the concentrations of NADP ÷ or its analogue from 4-250 ~ M and L-malate from 0.0430 mM. Concentrations of the other components in the assay mixture were held at saturation. Inhibition studies were performed in a similar

71

~

manner by varying the concentration of NADP + (from 10-30 ~ M ) while the pyridine nucleotide inhibitor was held at several fixed concentrations around its inhibition constant. Protein measurement. Total protein was deter-

mined after Lowry et al. (1951), or alternatively, by the method of Bradford (1976). BSA was used as standard. Data analysis. Reciprocal initial velocities were

plotted versus reciprocal substrate concentrations. Data were fitted to Eq. (1) V=

Vmax" [A]. [B] K d • K B + KmA[B] + KBm"[A] + [A]. [B] (1)

where [A] and [B] represent the concentrations of the pyridine nucleotide and L-malate, respectively. K~, K B and K d are the Michaelis constants for A and B and dissociation constant for the binary complex of the pyridine nucleotide, respectively. Vmax represents the maximum velocity of the reaction. " Initial velocity data exhibiting linear competitive and non-competitive inhibition were fitted to Eqs. (2) and (3), respectively, Vmax • [A] v = KmA(1 + [I]/K~) + [A] Wmax " [ A ]

v = K~(1 + [I]/K~s) + [A](1 + [I]/K.)

(2)

(3)

where [A] is the concentration of NADP ÷, [I] is the concentration of the inhibitors and Kis and Kii are the apparent inhibition constants associated with the effect of the inhibitor on the slopes ( K i s ) and intercepts ( K i i ) o f double-reciprocal plots (Cleland 1963). Theoretical calculations. MOPAC ver. 5 (for the IBM 9370-90 computer) (Stewart 1989) program was used. The PM 3 hamiltonian was employed in calculations of heats of formation and optimization of geometries. PRECISE and ANALYT keywords were used to ensure a high precision of calculations. Two sets of compounds were used as models of

R1

I

I:12 Fig. 1. Compounds used as models of NADP + for theoretical calculations.

NADP + and its analogue modified in nicotinamide moiety: R 1 = C(O)NH 2, C(S)NH2, NH2, C(O)CH 3 (Fig. 1). For the first s e t R 2 = C H 3. For this group, calculations were made for both the oxidized and reduced form of the coenzyme. In a second set of calculations, the R 2 substituent was expanded to phosphoribose to verify the simplification made in the first set.

Results To reveal the role of different parts of the NADP + molecule in the formation of a functionally competent complex with malic enzyme, the inhibitory properties of various analogues were tested. The compounds studied are shown in Fig. 2. The NADP + molecule can be divided into four regions, as illustrated in Fig. 3. Region 1 is the nicotinamide ring which is connected to the adenine structure (region 2) by region 4. Region 3 is the phosphate group that sterifies the ribose moiety. Region 4 is the two ribose rings and the pyrophosphate bridge that connects them. N A D P + analogues and fragments as inhibitors

Several NADP + analogues and fragments containing the adenine end have been tested as inhibitors of maize leaf malic enzyme. NHDP +, APADP+, AADP+, SNADP+, ~ NADP+, 2'3'NADPc +, 2'3'AMPc and 2'AMP were found to be inhibitors. The inhibition patterns were linear competitive with respect to NADP + for these analogues (Table 1). A typical result obtained with E NADP + is shown in Fig. 4a. The fragments containing the adenine end (3'AMP, 5'AMP, 3'5'AMPc, ADP, ATP, A and 3'NADP + and N A D +) exhibited linear non-competitive in-

72

R1

Table 1. Inhibition constants (K~) and pattern for the inhibition of maize malic enzyme by several NADP ÷ analogues

R2 O

O

II

I!

O-

OH

~/0~

OH

O I

O= P-OH I OH

R1

R2

NADP ÷

C(O)NH 2

Aden ine

SNADP+

C(S)NH 2

Adenine

AADP +

NH 2

Adenine

A PA DP ÷

C(O)C H 3

A d e n ine

NHDP +

C(O)N H 2

Hypoxanthine

ENADP +

C(O)N H 2

E t h e n o a d e n ine

Inhibition patterna

Kis (/x M)

NHDP ÷ APADP + AADP ÷ SNADP ÷ E NADP + 2'3'NADPc + 3'NADP ÷ NAD ÷ 2'AMP 2'3'AMPc 3'5'AMPc 3'AMP 5'AMP 5'ADP 5 'ATP A

C C C C C C NC NC C C NC NC NC NC NC NC

0.58 0.66 0.62 0.26 1.Ol 1.34 56.70 577.00 1.85 0.89 5.33 5.80 5.99 7.50 5.79 9.12

K, (/~ M)

53.07 558.00

5.93 5.62 6.03 8.05 5.67 9.87

aC: competitive inhibition. NC: non-competitive inhibition.

Fig. 2. Analogues of NADP +.

il

Compound

h i b i t i o n w i t h r e s p e c t to N A D P + ( T a b l e 1). T h e p a t t e r n o b t a i n e d w i t h 3 ' N A D P ÷ is i l l u s t r a t e d in Fig, 4b. #

N A D P ÷ analogues as alternative c o e n z y m e s

2

NH 2

Among the competitive inhibitors tested, several a r e active as a l t e r n a t i v e c o e n z y m e s . E N A D P ÷, N H D P + a n d A P A D P + e x h i b i t e d a p p a r e n t Vmax a n d K m v a l u e s s i m i l a r to t h o s e for t h e n a t u r a l c o e n z y m e while 2 ' 3 ' N A D P c + e x h i b i t a K m v a l u e larger than those of the other alternative coenz y m e s . K i n e t i c p a r a m e t e r s a r e listed in T a b l e 2.

Semiempirical results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . . . . O" . . . . . . . . . . . . . . . . . '........... 4' II

/

to

CH,TO- P-O- P - O - C I ' ~ O-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

OH

OH 0p ...........

q .........

O=P-OH I

OH

3

Fig. 3. The various regions within the molecule of NADP +.

T h e o r e t i c a l c a l c u l a t i o n s w e r e p e r f o r m e d with m o d e l s o f N A D P +, t h e n a t u r a l c o e n z y m e o f m a l i c e n z y m e , a n d S N A D P +, A A D P + a n d A P A D P ÷, a n a l o g u e s with s t r u c t u r a l c h a n g e s in t h e p y r i d i n e ring (Fig. 2). G e o m e t r y , c h a r g e distributions, dipole moments, ionization potentials a n d h e a t s o f f o r m a t i o n h a v e b e e n d e t e r m i n e d . R e s u l t s for t h e set o f c o m p o u n d s (Fig. 1) w h e r e t h e R 2 s u b s t i t u e n t was a p h o s p h o r i b o s e a r e s u m m a r i z e d in T a b l e 3. N o m e a n i n g f u l diff e r e n c e s in results b e t w e e n t h e two sets ( R 2 = CH 3 and R 2 = phosphoribose) were observed.

73 I

I

0.2

}

I

0.0

'

'

b

'7

.£ 0.2 ,¢[

0.O

- 0.1

0.O

O,1

IN ADP ÷] -1 (uM).~ Fig. 4. Double-reciprocal plot of malic enzyme activity versus NADW concentration (a) in the absence (O) and in the presence of 1/xM (O), 2/~M (A) and 3 IxM (A) ~NADP+; (b) in the absence (O) and in the presence of 25/zM (O), 50/zM (A) and 75 p.M (&) of 3'NADP ÷ at pH 8.

Table 2. Kinetic parameters for NADP + analogues as coenzymes for NADP ÷ malic enzyme from maizea

Discussion

Nucleotide

T h e initial-velocity d a t a i n d i c a t e that A P A D P ÷, E N A D P ÷ a n d N H D P ÷ are a l t e r n a t i v e coenz y m e s for this e n z y m e . T h e r e is less t h a n a t h r e e f o l d v a r i a t i o n in the values of the m a x i m u m velocities or Wmax/K m values for the four n u cleotides. 2 ' 3 ' N A D P c ÷, in which the third phosp h a t e of the c o e n z y m e is cyclized, can act as c o e n z y m e with a n o r m a l Vmax, b u t its K,~ v a l u e is m o r e t h a n a n o r d e r of m a g n i t u d e larger t h a n those for the a l t e r n a t i v e c o e n z y m e s with a n uncyclized T - p h o s p h a t e group.

NADW ~NADP + APADP + NHDP + 2'3'NADPc*

Kinetic parameters V~x (/zmol/min. rag)

Km Ab (/zM)

V/K~ (/zM)

K~, b

30.9 26.2 17.0 17.9 10.0

8.60 11.30 9.53 10.50 135.00

3.59 2.31 1.78 1.70 0.07

120 220 180 155 200

aVmax and K m w e r e determined by double-reciprocal plot. bKm A and KBmare the Michaelis constants for the pyridine nucleotide and L-malate, respectively.

74 Table 3. Calculated geometries and atomic charges of the atoms of the carboxamide of the nicotinamide ring for N A D P ÷ analogues Model

Angle

A t o m i c charges

ip c ev

NADP ÷ SNADP ÷ AADP ÷ APADP ÷

~ba

Ob

C

51 49 -56

180 177 174 176

0.24 -0.14 -0.31

O/S

N/C

-0.32 -0.14 --0.25

-0.04 0,21 0.17 -0.16

C4

0.02 0.00 -0.08 0.03

13.1 11.8 12.7 14.0

a~b - angle defining torsion of carbonyl oxygen out of the ring plane. b O - torsional angle between pyridine ring and ribose oxygen. tiP - ionization potential.

Thus, an intact adenine structure (region 2, Fig. 3) is not essential for activity. N H D P ÷ and E N A D P ÷ (Fig. 2) bind to the enzyme with approximately the same affinity as the natural coenzyme. However, the lower activities of these compounds suggest that interactions with the adenine ring are important for optimal orientation of the pyridine nucleotide for catalysis. In contrast, most structural changes at the nicotinamide end result in a loss of activity. The natural coenzyme and the alternative ones have a carbonyl group of the nicotinamide carboxamide at region 1 (Fig. 3). Ionization potentials for N A D P ~ and APADP ÷ were larger than those of the inhibitors (Table 3). Substitution of the carbonyl oxygen for a sulphur atom to give SNADP ÷ (Fig. 2) or removal of the carbonyl group to give A A D P ÷ (Fig. 2) leads to a change of the coenzyme activity. Therefore the carbonyl group of the nicotinamide carboxamide seems to be essential in the hydride-transfer reaction. Xray crystallography and Raman spectroscopy indicate that the carboxamide of the nicotinamide makes three hydrogen bonding contacts with dihidrofolate reductase (Filman et al. 1982). If this is important in orienting the nicotinamide ring in the binding site, then modifications which drastically affect the hydrogen binding ability of this group, as in APADP ÷ and SNADP ÷, should lead to an altered orientation of the ring. The proton chemical shifts indicate that the mode of binding of SNADP ÷ is quite different from that of N A D P ÷, while that of APADP ÷ is less so. This is reflected in their binding to the enzyme (Hyde et al. 1980a,b). The charge density on the Ca atom of the

pyridine ring is the major factor that governs the reactivity of the compounds studied. Presumably, the transition state for the nucleophilic attack by the reductant has a carbonium ion-like structure (Grau 1982). At the 3-position of the pyridine ring, a group with electron-withdrawing power sufficient to activate the 4-position can result in an active analogue (Anderson 1982). Table 3 shows that the charge density on the C a atom is positive for NADP ÷ and APADP ÷. Both inhibitors exhibit a negative partial charge on C 4 (the calculated value for SNADP ÷ is -0.002), which probably makes them resistant to hydride attack. The ribose moiety (region 4, Fig. 3) of the nicotinamide mononucleotide part is not essential for activity. The oxidation of NADP ÷ results 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. The oxidized NADP ÷ is active and binds pigeon NADP ÷ - m a l i c enzyme (Chang and Huang 1979). Changes in the position of the 2'-phosphate group (region 3, Fig. 3) are of importance for the binding of NADP ÷ since analogues which lack the 2'-phosphate group are bound more weakly by malic enzyme. The transference of the 2'phosphate group to the 3'-position results in a non-competitive analogue. However, the analogues and fragments tested with a 2'3'-phosphate group are competitive with respect to NADP-. Thus, the 2'-phosphate group is essential for nucleotide binding to many enzymes. For dihidrofolate reductase the phosphate group forms hydrogen bonds and salt bridges with

75 many aminoacid residues of this enzyme (Stone et al. 1984). In addition, studies using 31p-NMR spectroscopy with isocitrate dehydrogenase strongly implicate the 2'-phosphate group (Mas and Colman 1984). These results suggest that the NADP* molecule has two important regions for malic enzyme activity. The carboxamide carbonyl group of the nicotinamide moiety is crucial for coenzyme activity, whereas the 2'-phosphate group is important for nucleotide binding to the enzyme.

Acknowledgements This work was supported in part by a cooperative research grant from the Consejo Nacional de Investigaciones Cient/ficas y T6cnicas (CONICET, Argentina) and the National Science Foundation as well as by grants from TWAS to CSA (BC 90-024) and from the Commission of the European Communities (DG XII) to CSA, from NSF to MHO (INT-8901716) and from NIH to MHO (GM 43043). CPS is a Predoctoral Fellow of CONICET and CSA is an Investigator Career Member of the same institution.

References Anderson BM (1982) The pyridine nucleotide coenzymes. In: Everse J, Anderson B and Yon Ks (eds) pp 91-133. Academic Press, New York Andreo CS, Podestfi FE and Iglesias AA (1990) Current research in photosynthesis. In: Baltscheffsky M (ed) Sub-strate Binding to NADP-Malic Enzyme from Maize Leaves as Determined by Intrinsic Fluorescence Quenching, Vol. IV, pp 537-540. Kluwer Academic Publishers, The Netherlands Asami S, Inoue K, Matsumoto K, Murachi A and Akazawa T (1979) NADP*-malic enzyme from maize leaf: purification and properties. Arch Biochem Biophys 194:503-511 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 Chang GG and Huang TM (1979) Oxidized NADP as a potential active-site directed reagent of pigeon liver malic enzyme. Biochem Biophys Res Commun 86:829-836 Cleland WW (1963) The kinetics of catalysed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochem Biophys Acta 6:173-187

Drincovich MF, Andreo CS and Iglesias AA (1990) Calcium ion interaction with NADP-malic enzyme from maize leaves. Plant Physiol Biochem 28 (I): 43-48 Edwards GE and Huber SC (1981) The C a pathway. In: Hatch MD and Boardman NK (eds) The Biochemistry of Plants. A Comprehensive Treatise, Vol. 8, pp 237-281. Academic Press, New York Filman DJ, Bolin JT, Mattews DA and Kraust J (1982) Crystal structures of Escherichia coli and actobacillus casei dihydrofolate reductase refined at 1.7 ~ resolution. J Biol Chem 257:13663-13672 Frenkel S (1975) Regulation and physiological functions of malic enzymes. Curr Topic Cell Regul 9:57-181 Grau UM (1982) The pyridine nucleotide coenzymes. In: Everse J, Anderson B and Yon Ks (eds) pp 135-187. Academic Press, New York Hsu RY (1982) Pigeon liver malic enzyme. Mol Cell Biochem 43:3-26 Hsu RY and Pry TA (1980) Kinetic studies of the malic enzyme of pigeon liver. 'Half of the sites' behavior of the enzyme tetramer in catalysis and substrate inhibition. Biochemistry 19:962-968 Hsu RY, Mildvan AS, Chang GG and Fung CC (1976) Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn 2÷. J Biol Chem 251:6574-6583 Hyde El, Birdsall B, Roberts GCK, Feeney I and Burgen ASV (1980a) Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase. Biochemistry 19:3738-3746 Hyde EI, Birdsall B, Roberts GCK, Feeney I and Burgen ASV (1980b) Phosphorus-31 nuclear resonance studies of the binding of oxidized coenzymes to LactobaciUus casei dihydrofolate reductase. Biochemistry 19:3746-3754 Iglesias AA and Andreo CS (1989) Purification of NADPmalic enzyme and phosphoenolpyruvate carboxylase from sugarcane leaves. Plant Cell Physiol 30:399-406 Iglesias A A and Andreo CS (1990a) Kinetic and structural properties of NADP-malic enzyme from sugarcane leaves. Plant Physiol 92:66-72 Iglesias AA and Andreo CS (1990b) NADP-malic enzyme from sugarcane leaves: kinetic properties of its different oligomeric structures. Eur J Biochem 192:729-733 Jawali N and Bhagwat AS (1987) Presence of essential histidine residues in NADP-malic enzyme from maize. Phytochemistry 26:1859-1862 Lee HJ and Chang GG (1987) Interactions of NADPanalogues and fragments with pigeon liver malic enzyme. Biochem J 19:962-968 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 Mas MT and Colman RF (1984) Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP-specific isocitrate dehydrogenase. Biochemistry 23: 1675-1683 Schimerlik MI, Grimshaw CE and Cleland WW (1977) pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme. Biochemistry 16:571-576

76 Slack CR and Hatch MD (1967) Comparative studies on the activity of carboxylases and other enzymes in relation to the new pathway of photosynthetic carbon dioxide fixation in tropical grasses. Biochem J 103:660-665 Stewart JJP (1989) Quantum Chemistry Program Exchange 581 Stone SR, Mark A and Morrison JF (1984) Interaction of

analogues of nicotinamide adenine dinucleotide phosphate with dihydrofolate reductase from Escherichia coli. Biochemistry 23:4340-4346 Vernon CM and Hsu RY (1983) Pigeon liver malic enzyme. Involvement of an arginyl residue at the binding site for malate and its analogs. Arch Biochem Biophys 225: 296305