Conformation of NAD+ Bound to Allosteric L-Lactate Dehydrogenase Activated by Chemical Modification

Koide, Shohei; Yokoyama, S; Matsuzawa, Hiroshi; Miyazawa, Tatsuo; Ohta, Takao

doi:10.1016/s0021-9258(18)81845-2

Cited by 15 publications

(11 citation statements)

References 15 publications

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“…We had already demonstrated that the allosteric property of the enzyme can be manipulated with chemical and genetical modifications (Taguchi et al, 1984;Matsuzawa et al, 1988). We had also identified the conformational change of the enzyme-bound NAD"1" upon the binding of FBP (Machida et al, 1985) and upon chemical modification (Koide et al, 1989) by means of NMR spectroscopy. Therefore, tcLDH is advantageous for demonstrating the conformational equilibrium of the catalytic site.…”

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confidence: 99%

“…NMR Spectroscopy. A protein solution in buffered 2H2G containing 50 mM [2Hu]Tris (MSD Isotopes)-2HCl (pH* 7.1 at 25 °C, direct meter reading) and 1 M KC1 was prepared by repeated ultrafiltration, as described previously (Koide et al, 1989). The final concentrations of the enzymes were between 0.14 and 0.30 mM expressed as the concentration of subunit.…”

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confidence: 99%

“…NAD+ was added to the solution so that the molar ratio of NAD+/LDH was kept at 10. NMR measurements were performed with a Bruker AM600 spectrometer with an Aspect3000 computer, essentially following the previous procedures (Koide et al, 1989). NMR spectra were recorded at 40 °C, with 16K data points for a 9.6-kHz spectral width.…”

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confidence: 99%

“…The accuracy of the initial slopes was within ±20%. Interproton distances were determined as described previously (Clore & Gronenborn, 1983; Koide et al, 1989).…”

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confidence: 99%

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Conformational equilibrium of an enzyme catalytic site in the allosteric transition

Koide¹,

Yokoyama²,

Matsuzawa³

et al. 1992

Biochemistry

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The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.

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confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

“…The accuracy of the initial slopes was within ±20%. Interproton distances were determined as described previously (Clore & Gronenborn, 1983; Koide et al, 1989).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Conformational equilibrium of an enzyme catalytic site in the allosteric transition

Koide¹,

Yokoyama²,

Matsuzawa³

et al. 1992

Biochemistry

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“…Transferred NOE (TRNOE) measurements give information on the conformation of the bound ligand (Albrand et al, 1979;Clore & Gronenborn, 1982; Behling et al, 1988) . The TRNOE method has been applied to investigations on nucleotide-enzyme interaction (Cayley et al, 1979;Levy et al, 1983;Gronenborn et al, 1984a;Banerjee et al, 1985;Machida et al, 1985;Ferrin & Mildvan, 1985;Leanz & Hammes, 1986; Koide et al, 1989), peptide-phospholipid bilayer interaction (Wakamatsu et al, 1986(Wakamatsu et al, , 1987Milon et al, 1990), codon-anticodon interaction (Gronenborn et al, 1984b;Clore et al, 1984), and peptide-enzyme interaction (Clore et al, 1986;Meyer et al, 1988).…”

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confidence: 99%

Interaction mechanism between microtubule-associated proteins and microtubules. A proton nuclear magnetic resonance analysis on the binding of synthetic peptide to tubulin

Kotani¹,

Kawai²,

Yokoyama³

et al. 1990

Biochemistry

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An amino acid sequence essential for microtubule-associated proteins (MAPs) to bind to microtubules is presented [Aizawa et al. (1989) J. Biol. Chem. 264, 5885-5890]. A synthetic peptide of 23 amino acid residues which corresponded to the sequence [tubulin binding peptide (TBP)] was active in binding to tubulin and inducing its assembly. The TBP-tubulin interaction mechanism was analyzed by proton nuclear magnetic resonance spectroscopy as a simplified model for MAP-microtubule interactions. Intraresidue transferred nuclear Overhauser effects (TRNOEs) of TBP in TBP-tubulin mixtures were analyzed, and strong binding of two Val and two Lys residues of TBP to tubulin was detected. Among the sharply peaked signals from tubulin aromatic residues, those due to Tyr ring protons broadened upon mixing with TBP, suggesting the involvement of Tyr residue(s) in the binding with TBP. Irradiation of the tubulin Tyr protons resulted in an intermolecular TRNOE at TBP methyl proton resonances. Evidently, hydrophobic interactions between Val and Tyr residues are important for the binding of TBP to tubulin. Hydrophobic interactions have not been taken into account previously in the widely accepted electrostatic model for the binding of MAPs to microtubules.

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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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Conformation of NAD+ Bound to Allosteric L-Lactate Dehydrogenase Activated by Chemical Modification

Cited by 15 publications

References 15 publications

Conformational equilibrium of an enzyme catalytic site in the allosteric transition

Conformational equilibrium of an enzyme catalytic site in the allosteric transition

Interaction mechanism between microtubule-associated proteins and microtubules. A proton nuclear magnetic resonance analysis on the binding of synthetic peptide to tubulin

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

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