Alexander N. Malyan

Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF(1)) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF(1) with Mg(2+) produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k(-2)(ADP)=1.5 x 10(-1) min(-1) and k(-2)(ATP) congruent with 10(-3) min(-1), respectively. As follows from the study, the noncatalytic sites of CF(1) are not homogeneous. One of them retains the major part of endogenous ADP after CF(1) precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.

Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 μM) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.

The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca2+- and Mg2+-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca2+-dependent ATPase without changing the apparent Michaelis constant (Кm), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg2+-dependent reaction, the presence of sucrose increased Кm without changing the maximal rate that is many-fold as high as that of Ca2+-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg2+-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

IDENTIFICATION OF SURFACE EXPOSED AMINO ACIDS OF ISOLATED AND MEMBRANE-BOUND CF1 BY PROTEASE ACCESSIBILITY

In order to compare surface-exposed amino acids in isolated and membrane-bound CF1 the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF1 the N-terminal region of the α subunit was found to be highy sensitive to proteases; the accessible peptide bonds included αE17-G18, αR21-E22, αE22-V23, and αK24-V25. Additional protease-attacked bonds in α subunit were αS86-S87, xαE125-S126. and αR127-L128. In the β subunit of isolated CF1 the bonds βL14-E15 and βV76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF1 computed after the crystal structure of mitochondrial F1 by S. Engelbrecht (1997). In membrane bound CF1 the primarily accessible peptide bond of the N-terminal domain of α is αR21-E22. After this bond is cleaved by trypsin, the αK24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds αR14O-S141 and αG16O-R161 are cleaved. According to the Engelbrecht model αG16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF1. The β subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF1. In the β subunit of membrane-bound CF1 a papain-sensitive bond βG102-G103 was identified. The results indicate major structural alterations when CF1 is extracted from the CF0CF1 complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between γC199 and γC205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular ααG160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.

Examination of catalytic activity of the β subunit isolated from chloroplast coupling factor 1

Abstract A simple and rapid procedure has been developed to isolate the subunit from chloroplast coupling factor 1 involving anion exchange HPLC. The β subunit was mainly in the monomeric form, along with some quantities of trimer and more complex aggregates of the protein. Decreasing pH down to neutral values or heating resulted in shifting the equilibrium in the medium to the formation of polymeric forms of the β subunit. By successive purification and fractionation of CF 1 and β subunit preparations, it was shown that the monomeric form of the β subunit catalysed ATP-ADP γ-phosphate exchange. However, it did not display ATPase or adenylate kinase activities. Aggregation of the β subunit was accompanied by irreversible inactivation of its ATP-ADP exchange activity.

Chloroplast ATP Synthase: Identification of Protease Accessible Sites in CF 1

ATP synthases of chloroplasts, mitochondria and bacteria couple ATP synthesis/hydrolysis with transmembrane proton translocation. They are comprised of the water soluble catalytic F1 and the membrane-embedded proton conducting F0. The structure of F1 section of mitochondrial ATP synthase has been resolved by X-ray analysis [1]. The 3D structure of spinach CF1 has been modelled according to the crystal structure of MF1 [2]. Functionally, however, CF1 is rather different from MF1. CF1 is a latent ATP-ase and requires artificial activation to display catalytic activity. Moreover it is much more effectively inhibited by excess Mg2+ than MF1. CF0CF1 is a latent enzyme, too, and is activated by the transmembrane proton gradient. Unlike the mitochondrial F0F1, CF0CF1 undergoes redox regulation. It may also be challenged if the F1 structure can be simply projected into the intact F0F1. Electron microscopic analysis of 2D crystals at least suggested that the gross structure of isolated CF1 may rather different from the structure of CF1 in the intact CF0CF1 complex [3]. The technique of protease accessibility has been used to study the surface topology of proteins. Amino acid residues at peptide bonds accessible to proteolytic enzymes are supposed to be located at the protein surface. Here we have used this approach to identify exposed peptide bonds in isolated and membrane-bound CF1. The goals of these studies were (1) to examine by spot-checks the topology of CF1 with regard to the 3D structure modelled according to the structure of MF1, (2) to examine which of the amino acids accessible in isolated CF1 show also protease accessibility in CF0CF1, (3) to study changes in the accessibility of the protein bonds induced by membrane energization.

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [14C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [14C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m−2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.