The effect of valine substitution for glycine in the dimer interface of citrate synthase from Thermoplasma acidophilum on stability and activity

2000-08-28
To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (K-m for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., V-max, K-cat and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 --> Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS. (C) 2000 Academic Press.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Suggestions

The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum
Kocabıyık, Semra; Russel, RJM; Danson, MJ; Hough, DW (Elsevier BV, 1996-07-05)
In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate s...
The low spin - high spin equilibrium in the S-2-state of the water oxidizing enzyme
Boussac, Alain; Ugur, Ilke; Marıon, Antoıne; Sugiura, Miwa; Kaila, Ville R. I.; Rutherford, A. William (Elsevier BV, 2018-05-01)
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S-0 to S-4) before water is oxidized and O-2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S-2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S-2(LS) and S-2(HS) is pH depend...
Investigating the malleability of RNA aptamers
İlgü, Müslüm; Lamm, Monica H.; Nilsen-Hamilton, Marit (Elsevier BV, 2013-09-15)
Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemic...
Amino acid substitutions in the subunit interface enhancing thermostability of Thermoplasma acidophilum citrate synthase
Erduran, I; Kocabıyık, Semra (Elsevier BV, 1998-08-19)
We have used citrate synthase from Thermoplasma (Tp.) acidophilum as a thermostable model system to investigate the role of hydrophobic interactions in dimer interface for maintaining high temperature stability. Three mutant enzymes were constructed by single amino acid substitutions in the interface helices: Ala97 --> Ser, Ala104 --> Thr, and Gly209 --> Ala. All of the mutations enhanced the thermostability of Tp. citrate synthase, while improving its catalytic properties (K-m, V-max, and specific activity...
Using artificially generated spectral data to improve protein secondary structure prediction from Fourier transform infrared spectra of proteins
Severcan, M; Haris, PI; Severcan, Feride (Elsevier BV, 2004-09-15)
Secondary structures of proteins have been predicted using neural networks from their Fourier transform infrared spectra. To improve the generalization ability of the neural networks, the training data set has been artificially increased by linear interpolation. The leave-one-out approach has been used to demonstrate the applicability of the method. Bayesian regularization has been used to train the neural networks and the predictions have been further improved by the maximum-likelihood estimation method. T...
Citation Formats
S. Kocabıyık, “The effect of valine substitution for glycine in the dimer interface of citrate synthase from Thermoplasma acidophilum on stability and activity,” BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, pp. 460–465, 2000, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/47279.