Some properties of the citrate synthase from the extreme halophile, Halobacterium cutirubrum

Abstract

1. Citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating), EC 4.1.3.7] was purified about 400-fold from the extreme halophile, Halobacterium cutirubrum, by a method involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and hydroxyapatite and gel filtration on Sephadex G-200. 2. The purified enzyme was best activated by high concentrations of KCl (3M); the chlorides of other cations and K+ salts of other anions (Br-, NO3-, SCN-) were less effective than KCl as activators. The enzyme was best stabilized by high concentrations of NaCl or KCl. Cold-lability was found in the presence of 3M-KCl, but not in the presence of NaCl at concentrations up to 5M. The results suggest that both the shielding of negative charges on the enzyme molecule and the stabilization of hydrophobic bonds by high KCl concentrations were required for maximum activity of the enzyme. 3. The double-reciprocal plots for acetyl-CoA or oxaloacetate at several concentrations of the co-substrate intersected at the abscissa in the presence of either KCl or NaCl, at either 1 or 3M. The Km for oxaloacetate increased about fivefold with the salt concentration, from 1 to 3M.

Effect of Salt on the Activity and Stability of Aspartate Aminotransferase from the Halophilic Archaebacterium Haloferax mediterranei

“…This differ ence indicates a higher sensitivity of hAspAT to changes in the concentration o f K + than to N a +. A similar behavior, although with higher Hill's coef ficients, was found with other halophilic enzymes, such as pyruvate kinase [20], isocitrate dehydro genase [21] and citrate synthase [22]. This different effect for K + and N a + agrees with the different concentrations observed in the cell for these ions [23,24], which suggests a regulation o f the activity by the ratio of concentrations o f these ions in the cell.…”

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 2. Inactivation, Dissociation and Unfolding at NaCl Concentrations below 2 M. Salt, Salt Concentration and Temperature Dependence of Enzyme Stability

“…In contrast to malate dehydrogenase (h) the inactivation of NADH: menadione oxidoreductase [17] and citrate synthase [18] from H. cutirubrium exhibit negative Arrhenius slopes at high salt concentration. Thus low temperature destabilized these halophilic enzymes.…”

23. The biology of Antarctic saline lakes

“…It seems that in order for enzymes to function in such high concentrations of salt they must be modified, one of the major alterations being a dramatic increase in the proportion of acidic amino acids (reviewed by Lanyi 1974). However, the change in structure of the halophilic bacterial enzymes appears to have made a number of them cold-sensitive (Lanyi 1974;Norberg et al 1973;Higa & Cazzulo 1975). This may be the factor which has prevented the halobacteria from colonizing Antarctic lakes.…”