An investigation of the contribution made by the carboxylate group of an active site histidine-aspartate couple to binding and catalysis in lactate dehydrogenase

Clarke, Anthony R.; Wilks, Helen M.; Barstow, David A.; Atkinson, Tony; Chia, William; Holbrook, J. John

doi:10.1021/bi00405a034

Cited by 103 publications

(84 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the other hand, the reverse reaction requires unprotonated His (acting as a base) and the affinity for malate was not affected by the replacement of Asp by Ala or Asn. Similar results have been obtained by Clarke et al [23] who have used site-directed mutagenesis to study the contribution of Asp to the active site of a bacterial lactate dehydrogenase (the catalytic mechanism of which is assumed to be similar to that of malate dehydrogenases). They concluded that the formation of the ternary complex E-NADH-pyruvate was destabilized by the deletion of Asp but that the formation of E-NAD-lactate was not affected by the mutation since His was not positively charged in the oxidation of lactate.…”

Section: Discussionsupporting

confidence: 80%

“…However, for sorghum and D201N-mutated NADP-MDHs, no effect of the pH was observed in the pH range tested, which suggests that pK, of His229 is very high (> 9). Such a high value of pKd was predicted for NADP-MDH by Jackson et al [24], in contrast with cytosolic NAD-MDH [25] and bacterial lactate dehydrogenase [23] for which a pK, value of about 7 was determined. In contrast, k,,, of the D201A mutant seemed to be controlled by two ionisable groups.…”

Section: Discussionmentioning

confidence: 80%

See 1 more Smart Citation

The Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair

Lemaire¹,

Miginiac‐Maslow²,

Decottignies³

1996

European Journal of Biochemistry

View full text Add to dashboard Cite

Plant chloroplastic NADP-nialate dehydrogenase is unique among malate dehydrogenases because of its reductive activation in the light and cofactor specificity. In this paper, the role of His229 in sorghum leaf protein has been investigated by site-directed mutagenesis. His229 was replaced by Asn and Gln, both mutations yielding an inactive protein. The role of a conserved Asp (Asp201) as a possible partner of His229 in catalysis has been studied by the same approach. Both Asp mutants (D201A, D201N) were only slightly active and were essentially characterized by a dramatically increased K, for oxaloacetate (45 -SO-fold). pH dependence of catalytic rates revealed differences between the two Asp mutants. These results demonstrate that, in sorghum leaf NADP-dependent malate dehydrogenase, His229 is involved in catalysis in interaction with Asp201.Keywords: active site ; His-Asp pair; site-directed mutagenesis ; malate dehydrogenase.Malate dehydrogenase (MDH) is widely represented in the living kingdom, being found in every bacteria, yeast, animal and plant where it has been looked for [I, 21. In eukaryotes, this enzyme is present in the cytoplasm and in the mitochondria, both isoforms using NAD(H) as a cofactor [l]. The mechanism of the catalytic process occurring in cytoplasmic and mitochondrial MDHs has been known for a long time and the presence of a histidine at the active site of those proteins has been demonstrated by chemical derivatization [3 -51. Later on, the determination of their crystallographic structures highlighted the importance of an Asp residue, in interaction with the catalytic His [6, 71. More recently, the presence of this His/Asp pair was also found in the crystal structure of bacterial NAD-dependent MDH (NAD-MDH) from Escherichia coli [S] and Thermus fluvus [9]. However, no biochemical experiment has been performed to confirm the involvement of an Asp in the catalytic mechanism of NAD-MDH.In higher plants, a third isoform of malate dehydrogenase has been identified in another compartment of the cell, the chloroplast [ 101. This enzyme, NADP-dependent MDH (NADP-MDH), is a key catalyst in the photosynthetic CO, fixation pathway of C, plants [lo]. It uses NADPH as a coenzyme and is highly peculiar because of its light-modulated activity. Indeed, NADP-MDH is inactive in the dark and activated by light via the ferredoxin -thioredoxin system [ll]. Recently, we demonstrated that chloroplastic NADP-MDH also contains a His at the active site (H229 in sorghum) [12]. Sequence alignments indicated that this residue corresponds to the active-site His identified in NAD-MDHs (Fig. 1). In this paper, two mutant proteins were studied in which His229 was replaced by Asn (H229N Gln (H229Q). Moreover, the Asp interacting with the catalytic His in NAD-MDHs is strictly conserved in NADP-MDHs (Asp201 in sorghum protein). We report here our investigations on the role of this putative catalytical Asp in NADP-MDH and the interaction between this residue and His229. Site-directed mutagenesis allowed the production o...

show abstract

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 80%

The Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair

Lemaire¹,

Miginiac‐Maslow²,

Decottignies³

1996

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…This suggests a different mechanism for controlling substrate specificity compared with L-lactate dehydrogenase. The pH dependence of pyruvate K, in D-lactate dehydrogenase is in marked contrast to the situation found in L-lactate dehydrogenase, where pyruvate binds strictly to the protonated form of an activesite His195 which ionises in the binary complex with a pK, of about 7.5 (Clarke et al, 1988). This difference in pH dependence of pyruvate K, indicates that there is an alternative mechanism for substrate binding in D-lactate dehydrogenase compared with L-lactate dehydrogenase, which involves either a different ionisable group or a change in the environment of the active site histidine residue such that it has a perturbed pK,.…”

Section: Discussionmentioning

confidence: 61%

“…In contrast, Bacillus stearothermophilus L-lactate dehydrogenase shows the reverse trend of a similar magnitude with a preference for 2-oxobutyrate (Hart et al, 1987) over oxalacetate (Clarke et al, 1987). It is relevant that the specificity of L-lactate dehydrogenase for oxalacetate can be enhanced considerably by engineering an arginine residue as a counter ion for the negatively charged carboxyl group (Clarke et al, 1988). Comparative analysis of substrate specificities of L-and D-lactate dehydrogenase suggests that the latter may have a positively charged amino acid residue in the vicinity of the active site which could interact with a negatively charged group at position C3 of the 2-0x0-acid substrate, i.e.…”

Section: Discussionmentioning

confidence: 98%

Cloning and overexpression of Lactobacillus helveticusd‐lactate dehydrogenase gene in Escherichia coli

Kochhar

Hottinger

Chuard

et al. 1992

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

NAD +-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220 -21 5 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267,, a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene.Both plasmids overexpressed D-hCtate dehydrogenase ( > 60% total soluble cell protein) and were stable in Escheriehia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillusplantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184,60 -661. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. cx2 dimeric subunit structure, isoelectric pH, K , and k,,, for pyruvate and other 2-0x0-acid substrates. The kinetic profiles of 2-0x0-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.

show abstract

“…Site directed mutagenesis studies on LDH and mMDH indicate that both enzymes have a histidine located in the active site which interacts with an aspartate, and have proton-donating and accepting capabilities [17,18]. Furthermore, a…”

Section: Introductionmentioning

confidence: 99%

Mechanistic Studies on Malate Dehydrogenase from Escherichia coli

Wright

Zhao

Rardin

et al. 1995

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

An investigation of the contribution made by the carboxylate group of an active site histidine-aspartate couple to binding and catalysis in lactate dehydrogenase

Cited by 103 publications

References 25 publications

The Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair

The Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair

Cloning and overexpression of Lactobacillus helveticusd‐lactate dehydrogenase gene in Escherichia coli

Mechanistic Studies on Malate Dehydrogenase from Escherichia coli

Contact Info

Product

Resources

About