Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants.

The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary molecular determinants.

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component. A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria. Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform. Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles. Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria. The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.

Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin.

Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria). With porin-containing yeast mitochondria it is shown that (i) a significant, saturable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism. This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast. With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion. The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase-yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems.

Localized firefly luciferase probes ATP at the surface of mitochondria.

The concentration of ATP generated by yeast mitochondria and consumed by yeast hexokinase was monitored using native firefly luciferase in solution, or recombinant luciferase localized at the surface of mitochondria. In the absence of hexokinase, both probes perform similarly in detecting exogenous or mitochondrially-generated ATP. The steady-state concentrations of ATP can be reduced in a dose-dependent manner by hexokinase. With hexokinase added in large excess, the localized probe reports substantial ATP concentrations while none is detectable by soluble luciferase. Thus, ATP accumulates near the membrane where it appears, relatively to solution, and vice versa for ADP. The extent of nucleotide gradients is shown to be correlated with the specific activity of oxidative phosphorylation and with the viscosity of the medium, but independent of the concentration of the organelles. A simple model involving diffusional restrictions is presented to describe this behavior. The metabolic and evolutionary implications of cellular catalysis limitation by physical processes are discussed.

Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: characterization using [32P]3'-O-(4-benzoyl)benzoyl ATP, a photoaffinity label.

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.

Hydrophobic docking: a proposed enhancement to molecular recognition techniques.

In the classical procedures for predicting the structure of protein complexes two molecules are brought in contact at multiple relative positions, the extent of complementarity (geometric and/or energy) at the surface of contact is assessed at each position, and the best fits are retrieved. In view of the higher occurrence of hydrophobic groups at contact sites, their contribution results in more intermolecular atom-atom contacts per unit area for correct matches than for false positive fits. The hydrophobic groups are also potentially less flexible at the surface. Thus, from a practical point of view, a partial representation of the molecules based on hydrophobic groups should improve the quality of the results in finding molecular recognition sites, as compared to full representation. We tested this proposal by applying the idea to an existing geometric fit procedure and compared the results obtained with full vs. hydrophobic representations of molecules in known molecular complexes. The hydrophobic docking yielded distinctly higher signal-to-noise ratio so that the correct match is discriminated better from false positive fits. It appears that nonhydrophobic groups contribute more to false matches. The results are discussed in terms of their relevance to molecular recognition techniques as compared to energy calculations.

Local probes and heterogeneous catalysis: a case study of a mitochondria-luciferase-hexokinase coupled system.

Biological systems are characterized by a high degree of structural organization. In the intracellular context, this introduces physical constraints which are not considered in the standard biochemical analysis of isolated systems, aimed towards mechanistic studies. A major challenge in cellular biology is thus to integrate the structural and mechanistic information and reach an adequate representation of the modes of operation in situ. We present an approach to this problem which takes advantage of a localized probe to study heterogeneous coupled system, as minimal models for cellular operation. The system consists of ATP production at the surface of mitochondria, and ATP consumption in solution by the hexokinase reaction. Soluble or biologically localized firefly luciferase is used to continuously monitor ATP concentration either in the bulk solution or at the surface of the organelle, respectively. The general system of a surface source and a bulk sink is mathematically modeled, and an analytic steady-state solution for local and bulk ATP is presented. The results are validated by experiment and differ from the expected behavior of an equivalent homogeneous system in solution. The model is further adapted to evaluate the effect of mixing. In addition, two limiting cases of heterogeneous distribution of hexokinase are analyzed, in which the soluble enzyme adsorbs non-specifically to mitochondria, or binds selectively to the site of ATP appearance on the membrane. The results are discussed in terms of their significance to the analysis of bulk measurements in vitro and their relevance to better description of cellular situations.

Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques.

A geometric recognition algorithm was developed to identify molecular surface complementarity. It is based on a purely geometric approach and takes advantage of techniques applied in the field of pattern recognition. The algorithm involves an automated procedure including (i) a digital representation of the molecules (derived from atomic coordinates) by three-dimensional discrete functions that distinguishes between the surface and the interior; (ii) the calculation, using Fourier transformation, of a correlation function that assesses the degree of molecular surface overlap and penetration upon relative shifts of the molecules in three dimensions; and (iii) a scan of the relative orientations of the molecules in three dimensions. The algorithm provides a list of correlation values indicating the extent of geometric match between the surfaces of the molecules; each of these values is associated with six numbers describing the relative position (translation and rotation) of the molecules. The procedure is thus equivalent to a six-dimensional search but much faster by design, and the computation time is only moderately dependent on molecular size. The procedure was tested and validated by using five known complexes for which the correct relative position of the molecules in the respective adducts was successfully predicted. The molecular pairs were deoxyhemoglobin and methemoglobin, tRNA synthetase-tyrosinyl adenylate, aspartic proteinase-peptide inhibitor, and trypsin-trypsin inhibitor. A more realistic test was performed with the last two pairs by using the structures of uncomplexed aspartic proteinase and trypsin inhibitor, respectively. The results are indicative of the extent of conformational changes in the molecules tolerated by the algorithm.

Targeting of cloned firefly luciferase to yeast mitochondria.

The firefly luciferase gene (luc) was fused to a 5' fragment of the 70-kDa protein gene (70K) from yeast. The fragment codes for the N-terminal putative signal sequence which targets and anchors the 70-kDa protein to the cytoplasmic side of the outer membrane in mitochondria. Two versions of the fusion gene, 70K[232]::luc and 70K[93]::luc (containing 292 and 93 5' codons from 70K, respectively), were constructed in a bacterial expression plasmid. Both the genes were expressed in Escherichia coli, and in both cases, bioluminescence activity was associated with the expression. The 70K[93]::luc gene was transferred to a yeast-bacteria shuttle vector used to transform Saccharomyces cerevisiae cells. As a control, the same strain was transformed with a plasmid including the original luc. With both transformants, bioluminescence activity was detected in intact cells and crude extracts. Upon growth on a nonfermentable carbon source and fractionation, the product of the fusion gene was associated mostly with mitochondria. In the control transformant, the product of luc was more delocalized. However, a significant amount remained associated with isolated mitochondria. No such spontaneous association of purified luciferase with wild-type mitochondria was observed in vitro. Trypsin treatment of mitochondria isolated from both transformed strains indicated that the fusion protein is anchored to the outer membrane and exposed to the medium while the unfused luciferase retained with the mitochondria is occluded in a compartment unaccessible to trypsin and released in the presence of detergent. The fusion protein retained the major catalytic properties of the parent firefly luciferase, as determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase.

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.

Source of rapidly labeled ATP tightly to non-catalytic sites on the chloroplast ATP synthetase.

Bound [32P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [32P]Pi. Tight binding of [32P]ATP occurred both during and after energization. Different classes of bound [32P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP. 1. The rates of formation and discharge of the rapidly labeled tightly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation. 2. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation. 3. Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non-catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.