Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Sakowicz, Roman; Kallwass, Helmut; Parris, Wendy; Kay, Cyril M.; Jones, J. Bryan; Gold, Marvin

doi:10.1021/bi00210a023

Cited by 13 publications

(10 citation statements)

References 33 publications

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“…No substitutions occur in the catalytic loop region (amino acids 96-113; ref. 22) or in substrate and cofactor binding residues found elsewhere in the sequence (22,(30)(31)(32). (Note that the residue numbering system used in this paper is based on the notothenioid consensus sequence except (29), the goby G. mirabilis (2), the barracudas Sphyraena idiastes and S. lucasana (3), the eelpout Austrolycos depressiceps, and the lampfish Lampanyctus ritteri (P.A.F.…”

Section: Collectionmentioning

confidence: 99%

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Fields

Somero

1998

Proc. Natl. Acad. Sci. U.S.A.

375

344

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To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A 4 (A 4 -LDH) from nine Antarctic (؊1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis-Menten constants (K m ) and catalytic rate constants (k cat ) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in k cat and K m are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A 4 -LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting k cat , we conclude that notothenioid A 4 -LDHs have adapted to cold temperatures by increases in f lexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperatureadaptive variation in K m and k cat . Changes in sequence that increase f lexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase k cat . However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high K m values relative to warmadapted orthologs.

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Section: Collectionmentioning

confidence: 99%

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Fields

Somero

1998

Proc. Natl. Acad. Sci. U.S.A.

375

344

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“…L-LDH has been found to function as an allosteric enzyme which is activated by fructose 1,6-diphosphate (FBP) or as a nonallosteric enzyme (11). Allosteric L-LDHs have been purified, characterized, and cloned from a variety of eukaryotes and prokaryotes, and their primary and tertiary structures have been extensively studied (5,6,14,29,42). Much less information is available about the structure and regulation of bacterial nonallosteric L-LDHs.…”

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confidence: 99%

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme

Savijoki¹,

Palva²

1997

Appl Environ Microbiol

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The Lactobacillus helveticus L-(؉)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5 end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative ؊10 regions, but no corresponding ؊35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the ؊10 region closest to the initiation codon. However, upstream of these regions additional putative ؊10/؊35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonallosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K m , and K cat for pyruvate were 0.25 mM and 643 s ؊1 , respectively.

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“…4(e)]. In the T state of the enzyme, consequently, Ile240 occupies the space where a substrate would bind, and Thr246, which plays an important role in the catalytic reaction of the enzyme,48, 49 is far from the substrate binding site. In the case of LCLDH, in contrast, the α1/2G helix is consistently kinked at position 234 between MR2 and MR3 (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

et al. 2009

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Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium.

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Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Cited by 13 publications

References 33 publications

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

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Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Cited by 13 publications

References 33 publications

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4orthologs of Antarctic notothenioid fishes

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4orthologs of Antarctic notothenioid fishes

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

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Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes