Vol. 189, No. 2, 1992 December 15. 1992

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THE IMPORTANCE OF ARGININE 102 FOR THE SUBSTRATE SPECIFICITY OF ESCHERICHIA COLI MALATE DEHYDROGENASE David J. Nicholls’, Julie Miller’, Michael D. Scawen’, Anthony R. Clarke’, J. John Holbrook2, Tony Atkinson’ and Christopher R. Goward”

‘Division of Biotechnology,

PHLS Centre for Down, Salisbury 2Molecular Recognition Biochemistry, University of Bristol, School

Received

October

26,

Applied Microbiology and Research, Porton SP4 OJG, UK Centre, Department of of Medical Sciences, Bristol BS8 lTD, UK

1992

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the 0 1992 Academic Press, Inc. formation of a salt bridge with a carboxylate group of oxaloacetate.

Understanding the features which control enzyme substrate specificity is of fundamental importance in biochemistry. Many enzymes show a high degree of specificity for small ligands regardless of the small number of potential sites for interaction. The types of interaction and the mechanisms by which enzymes discriminate between small ligands of similar structure is poorly understood. The work described here is part of a study to determine the factors controlling the molecular recognition of keto-acid substrates by Escherichia coli malate dehydrogenase (MDH). X-ray crystallography has shown that there is considerable structural identity between malate dehydrogenase and lactate dehydrogenases (LDH) [ 1,2]. The amino acid residues of MDH in this study are numbered according to the system described for the analogous enzyme LDH by Eventoff et al. [3]. The amino acid residue at position 102 was previously shown to ‘To whom correspondence should be addressed. Abbreviations: HP, hydroxypyruvate; KB, ketobutyrate; KC, ketocaproate; KG, ketoglutarate; KV, ketovalerate; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; OAA, oxaloacetate; NADH, nicotinamide-adenine dinucleottde; PP, phenylpyruvate; PYR, pyruvate; RBE, relative binding energy. 0006-291X/92 1057

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be of critical importance for conferring MDH activity on to an LDH enzyme framework [4]. The shift in specificity from pyruvate to oxaloacetate was effected by mutation of the I& gene to change the amino acid residue Q102R. The mutant enzyme was a highly specific catalyst for oxaloacetate and k,,,/K, was equal to that of wild-type LDH for pyruvate. The residue at amino acid position 102 is mostly Gln in LDHs, but is conserved as Arg in MDH. By performing the reverse operation with the structurally related MDH to create a mutant enzyme R102Q, we proposed to investigate the importance of the conserved R102 in conferring substrate specificity to an MDH enzyme framework. Materials and Methods E. coli was grown in 2xYT broth [2 % (w/v) tryptone, 2 % (w/v) yeast extract, 1% (w/v) NaCl] at 37°C. Ampicillin (100 pg ml-‘) was used where necessary to select for clones containing recombinant plasmids. Standard recombinant DNA techniques were used as described by Maniatis et al. [5]. E. coli TG2 (K12,(Zuc-pro), supE, thi, h.rdDSIF’, traD36, proA+B+, ZacIq, 1acZ Ml5 recA) was the host strain for recombinant plasmids and phage. Oligonucleotide site-directed mutations were introduced into the mdh gene by using the single primer extension method [6]. The entire nucleotide sequence was determined to check that only the desired mutation was incorporated following mutagenesis. The mutated gene was ligated as a 2.2 kb SphIIEcoRI fragment into pMTL23 [7l for expression of the mutant recombinant enzyme. Wild-type and mutant MDH were purified by the previously reported method [8]. The purity of enzyme preparations was assessed by SDS-PAGE. Protein concentrations were determined by using the Biuret method [9] with bovine serum albumin as the standard. Kinetic data were determined at 30°C in 50 mM-Tris/HCl buffer, pH 7.5, containing either 0.14 mM-NADH or 0.3 mM-oxaloacetate as the fixed substrates as appropriate. Kinetic constants for wild-type and mutant MDH were determined with various substrates over a concentration range from 0.005 to 50 mM as appropriate. The initial reaction rates were measured by following a change in AXan . Kinetic parameters were calculated by using a nonlinear regression data analysis program @nztitter, Elsevier-Biosoft). Keto-acid substrates used are presented in Table I. Results and Discussion The wild-type and mutant R102Q MDH were purified to specific activities of 1900 U mg-’ and 5 U mg-’ respectively. SDS-PAGE revealed a single protein band of MI 34000 for both enzymes. The ratio of k,,,/K, is related to the binding energy of an enzyme and substrate in the transition state by the equation RT In (kJK,> [lo]. The relative binding energy (RBE) indicates the effectiveness of an enzyme-substrate interaction with the advantage of only including the formation of catalytically productive complexes [ 111. Steady state kinetic data for wild-type and mutant MDH (‘Bible I) were used to calculate the RBE of substrates to the enzymes (Fig. 1). As the R102Q mutation does not have any consequence for the kinetics of binding to coenzyme (Table I), the dramatic reduction in specificity for oxaloacetate is as a result of a change in the substrate binding site. Wild-type MDH is highly specific for oxaloacetate as shown by the ratio of the kC,(Km for oxaloacetate and other keto-acid substrates (Fig. 2). The close structural homology between MDHs and LDHs suggests similarities in the active -sites and catalytic mechanisms in that the rate of catalysis is limited by movement of a mobile 1058

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able I Steady-state kinetic properties of wild-type and mutant MDH constant

Substrate

Wild-type MDH

Mutant MDH

HOOC-CO-R Ketoglutarate z 12 13’

R: -CH,-CH,-COOH

Oxaloacetate

0.04 931

R: -CH,-COOH

Pyruvate R: -CH

3

Hydroxypyruvate R: -CH;OH

Ketobutyrate

Ketovalerate R: -CH,-CH,-CH

ii77 257’

ii: 0.14

25 3.3 132

21 0.0065 0.31

83 3.3 40

68 0.07 1.03

3

Ketocaproate

5.2

23275000

FE 0.71

R: -CH2-CH,

NM NM

K 9.8 25 0.28 11.2

10 0.06 6

R: -CH,-CH2-CH2-CH,

Phenylpyruvate R: -CH2Q

NADH (oxaloacetate)

;08 13:3

5 1.5 300

:72 120’

0.05 749

0.03 0.13 4330

14980000 -

The units for Km, k and k IK were mM, s-’ and M-‘.s-! respectively. NM = not measurable.Where K ?&as too?%ge%o be measured,k lK was calculated from the slope of the saturation curve which was assumedto be linear at 1% &s&ate concentrations.

loop [4]. By analogy with LDH [4], when substrate and coenzyme enters the catalytic site, the mobile loop closes, water is displaced and reactive amino acid residues solvate the substrate. The environment in the active-site vacuole becomes hydrophobic and the guanidinium group of R102, which is located on the 13 residue loop [12], becomes buried. In the catalytically active complex between the wild-type MDH and oxaloacetate, the highly

basic and polar guanidinium

group of R102 is solvated and neutralised

by the

carboxylate anion of oxaloacetate and ion-charge complementarity is fulfilled (Fig. 3). The free energy is greatly reduced and a high binding affinity results. The hydration potential of Arg (-20 kcal/mol) is far greater than that of Gln (-9.4 kcal/mol) 1141 due to the greater

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12 0

10

0

,

-2

KG

0

1

OAA

PYR

HP

KEt

KV

KC

PP

a

0

1

1

KG

2

Keto-acid substrate

1 PYR

HP

KEJ

1 KV

KC

PP

Keto-acid substrate

1. Relative binding energy of substrateto wild-type and mutant enzymes. The kc,(Km ratio in Table I was converted to relative binding energy for various substrates by using the equation RT In k,,,lK, for wild-type (0) and mutant MDH (m).

Figure

Figure 2. Substrate specificity of wild-type and mutant enzymes with respect to oxaloacetate. The k,JK, values from lhble I are shown as the logarithm of the ratio of kc ,IK for oxaloacetate to k,,,lK, for substratefor the wild-type (black) and mutant enzymes (whiter

number of electronegative atoms in the guanidinium group than in the amide group. The high enthalpy of a buried charged interaction provides enough free energy to remove the water molecules from the hydration shells of R102 and the substrate. In this way water is removed from the active-site vacuole. Water cannot be efficiently

removed from the C4 carboxylatc

group of oxaloacetate without the strong carboxylate-guanidinium

interaction

with R102.

Exclusion of water from the active-site vacuole may be important for stability of the salt bridge between oxaloacetate and R102 in the wild-type MDH as electrostatic interactions are much stronger in an environment of low dielectric constant. As Arg has a greater number of bound

(a) Arginine

(b) Glutamine

3. The interaction of a carboxylate group with Arg and Gln. The interaction of the carboxylate group of the substrate is a salt bridge with (a) Arg in the wild-type enzyme, but only involves weak hydrogen bonding with (b) Gln in the mutant enzyme.

Figure

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water molecules than Gln, the number of water molecules displaced from the guanidinium group to the bulk phase is far greater than that displaced from the amide group, and therefore there is a larger entropic component in the Arg-oxaloacetate interaction. As in the case with the analogous LDH the binding of substrate in the active site of MDH will be largely entropy driven [15]. The R102Q mutation results in a decrease in selectivity for oxaloacetate over pyruvate of 8 orders of magnitude (Fig. 2). The RBE with oxaloacetate is considerably reduced from about 10.2 kcal/mol for the wild-type enzyme to about 3.3 kcal/mol for the mutant enzyme (Fig. 1). Oxaloacetate can hydrogen bond with R102Q MDH (Fig. 3) but the carboxylate anion is not neutralised as with R102 of the wild-type enzyme. The RBE of the wild-type enzyme with pyruvate is about -1.2 kcal/mol (Fig. 1). This negative value suggests that the enzyme actively selects against pyruvate; the methyl group of pyruvate, in place of the carboxylate group of oxaloacetate, cannot neutralise the buried charge of R102. Hydroxypyruvate is less actively selected against, with a RBE of about -0.7 kcal/mol (Fig. l), perhaps due to hydrogen bonding with R102 and thus partial neutralisation of the buried charge. The RBE of wild-type MDH with various monocarboxylic keto-acid substrates shows that increasing chain length, in the series of pyruvate, ketobutyrate, ketovalerate and ketocaproate, allows the substrate to be more readily bound. There may be less space available for water molecules in the active-site vacuole and hence less disruption of electrostatic interactions with other residues involved in substrate selection and catalysis. The RBE for the wild-type enzyme with phenylpyruvate is stabilised by 4.6 kcal/mol, compared with the value for pyruvate. This gain in RBE may be achieved by interaction of R102 with the phenyl group because aromatic rings can act as hydrogen bond acceptors arising from electrostatic and van der Waals’ interactions and contributes about 3 kcal/mol of stabilising enthalpy [13]. Although wild-type MDH is highly specific for oxaloacetate, there is no significant difference in RBE between wild-type and R102Q enzymes with ketoglutarate which has an additional methylene group, and so may be too large to be accomodated in the active site. The RBE of the mutant enzyme to monocarboxylic keto acids varies little with increasing chain length (Fig. l), as there is no buried guanidinium group to neutralise. The RBE of the R102Q mutant with phenylpyruvate is similar to that of pyruvate, which indicates there is no interaction of the aromatic ring with Gln. Wild-type MDH is highly specific for oxaloacetate (Figs. 1 & 2) and it is likely that the strong bifurcated bonding with a charged residue (R102) in the wild-type enzyme (Fig. 3) is crucial for the high degree of specificity towards oxaloacetate. Productive binding of pyruvate is prevented by the unfavourable entropic effect of burying the hydrated side chain of R102 in the active-site vacuole. In contrast to the situation with LDH [4], the mutation R102Q does not reverse the specificity from oxaloacetate to pyruvate. Indeed, in MDH the Arg residue actively selects against pyruvate as a necessity, because of the relatively high concentrations of pyruvate in viva. Lactate, the product of the LDH reaction, which would kill the cell if allowed to remain, is secreted by transport mechanisms. R102 may therefore be the result of a strong evolutionary pressure on an organism to develop a highly selective binding mechanism for oxaloacetate. 1061

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References :-

3: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Birktoft, J.J. and Banaszak, L.J. (1983) J. Biol. Chem. 258,472-482. Birktoft, J.J., Rhodes, G. and Banaszak, L.J. (1989) Biochemistry 28, 60656089. Eventoff, W., Rossman, M.G., Taylor, S.S., Torff, H.-J., Meyer, H., Keil, W. and Kiltz, H.H. (1977) Proc. Natl. Acad. Sci. USA 74,2677-2681. Wilks, H.M., Hart, K.W., Feeney, R., Dunn, C.R., Muirhead, H., Chia, W.N., Barstow, D.A., Atkrnson, T., Clarke, A.R. and Holbrook, J.J. (1988) Science 242, 1541-1544. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: a Zaboratory manual, Cold Spring Harbor, N.Y. Winter, G., Fersht, A.R., Wilkinson, A.J., Zoller, M. and Smith, M. (1982) Nature 299, 756-758. Chambers, S.P., Prior, S.E., Barstow, D.A., Minton, N.P. (1988) Gene 68, 139-149. Nicholls, D.J., Minton, N.P., Atkinson, T. and Sundaram, T.K. (1989) Appl. Microbial. Biotech. 31, 376-382. Gornall, A.G., Baradawill, C.J. and David, M.M. (1949) J. Biol. Chem. 177, 751766. Wilkinson, A.J., Fersht, A.R., Blow, D.M. and Winter, G. (1983) Biochemistry 22, 3581-3586. Hart, K.W., Clarke, A.R., Wigley, D.B., Waldman, A.D.B., Chia, W.N., Barstow, D.A., Atkmson, T., Jones, J.B. and Holbrook, J.J. (1987) Biochem. Biophys. Acta 914, 294-298. Hal!, M.D., Levitt, D.G. and Banaszak, L.J. (1992) J. Mol. Biol. 226, 867-882. Levitt, M. and Peru& M.F. (1988) J. Mol. Biol. 201, 751-754. Wolfenden, R., Andersson, L., Cullis, P.M. and Southgate, C.C.B. (1981) Biochemistry 20, 849-855. Hart, K.W., Clarke, A.R., Wigley, D.B., Chia, W.N., Barstow, D.A., Atkinson, T. and Holbrook, J.J. (1987) Biochem. Biophys. Res. Comm. 146, 346-353.

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The importance of arginine 102 for the substrate specificity of Escherichia coli malate dehydrogenase.

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The co...
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