Effect of pH, Mg²⁺, CO₂ and Mercurials on the Circular Dichroism, Thermal Stability and Light Scattering of Ribulose 1,5-Bisphosphate Carboxylases from Alfalfa, Spinach and Tobacco

Abstract

Circular dichroism, differential scanning calorimetry and light-scattering measurements of ribulose 1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from alfalfa, spinach and tobacco show: a) The conformation and thermal stability of the native carboxylases are sensitive to changes in pH and to activation of the enzyme with Mg2e and CO2. The helical content, denaturation temperature (Td) and specific enthalpy of denaturation (Aq) decreased with increase in pH. Addition of Mg2e and CO2 at pH 9 increased Td by 4 to 5 C; at pH 7.5 the changes in Td were smaller. b) Addition of mercurials produced changes in conformation and thermal stability. The decrease in helical content of the enzymes with increase in pH was enhanced by the addition of p-chloromercuribenzoate. At pH 9, addition of p-chloromercuribenzoate or of 1-(3-(chloromercuri)-2-methoxypropyl)urea decreased Td by 11.4 to 20.2 C and Aq by 2.1 to 2.8 calories per gram. c) The spinach carboxylase undergoes the largest and the tobacco the smallest changes in conformation and thermal stability upon change in pH or treatment with mercurials. d) The calorimetric data suggest that the large and small subunits are heat denatured independently but at the same temperature. e) Light scattering measurements at pH 9 of p-chloromercuribenzoate treated tobacco enzyme showed that there is no dissociation into subunits upon heating to temperatures greater than Td. A 'ball and string' model for the carboxylase molecule is proposed to reconcile independence of subunit denaturation with apparent strong interactions between subunits.Ribulose 1,5-bisphosphate carboxylases (EC 4.1.1.39) from higher plants like alfalfa, spinach and tobacco have similar molecular weights (16,27), amino acid and subunit compositions and sequences (16,19,21), but show distinct differences in enzyme activity and crystallizability. The specific activity of the spinach enzyme (23, 29) is about three times that of the alfalfa enzyme (27) and six times that of the tobacco enzyme (6, 18). Carboxylases from different tobacco species show differences in their amino acid composition but have the same specific activity (31). and four small spherical masses. Such a molecule should have minimum contact between the subunits and be readily dissociable into its subunits. However, in most cases, strong denaturants are required to separate the subunits and the isolated subunits are denatured on separation (14). Although Nishimura et al. (20) reported that treatment with p-chloromercuribenzoate at pH 9 dissociated the spinach enzyme, light scattering measurements (27) showed that the same procedure did not dissociate the alfalfa enzyme.Results of CD, differential scanning calorimetric and light scattering measurements of the effects of pH, Mg2+ and CO2, and mercurials on conformation, thermal stability and subunit cooperativity of the three carboxylases are presented in this report. In general, the measured properties of the three carboxylases are similar. There are small but significant differences in secondar...

Rubisco Adaptation to Low Temperatures: A Comparative Study in Psychrophilic and Mesophilic Unicellular Algae

“…Our data on the C. reinhardtii RUBISCO are in good agreement with those obtained by Chen et al (1993). In the mesophilic alga, as in spinach, tobacco, and alfalfa (Tomimatsu andDonovan 1981, Eckardt andPortis 1997), RUBISCO is a remarkably stable enzyme. The ''cold'' RUBISCOs analyzed in this work present an increased thermosensitivity when compared to the C. reinhardtii enzyme.…”

Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase

“…Activation by CO2 and metal ions with formation of a ternary complex is a property common to all rubiscos regardless of their taxonomic origin (Lorimer & Maziorko, 1983;Andrews & Lorimer, 1987). Interestingly, addition of Mg2+ and CO2 enhances the thermal stability of the hexadecameric rubisco without any effect on the subunit cooperativity (Tomimatsu & Donovan, 1981). It was also recently found that pretreatment of the enzyme with Mg2+ and CO2 prevents the inactivation that is known to occur in spinach rubisco upon storage of dilute solutions in the cold (Heuer & Portis, 1990).…”

Heat Denaturation Profiles of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) and Rubisco Activase and the Inability of Rubisco Activase to Restore Activity of Heat-Denatured Rubisco

“…lA), suggesting that sulfhydryl oxidation may play a role in heat denaturation in the absence of DTT. The stability of Rubisco is not limited to the spinach enzyme, since high denaturation temperatures were reported for Rubisco from tobacco and alfalfa as well as spinach (Tomimatsu and Donovan, 1981).…”