The continuously evolving glossary of terms - Claude Aflalo Under

[32P]3'-O-(4-Benzoyl)benzoyl ATP. Benzophenone derivative of ATP. A powerful photoaffinity labeling reagent for detection of ATP binding proteins, and characterization of the binding sites. Irradiation of a preformed BzATP-protein complex with UV light (360 nm) results in covalent derivatization of the protein which can be resolved by SDS-PAGE and detected by autoradiography.

The phenomenon in which a metabolite 'I', product of an enzyme 'E1', is directly transferred to the next enzyme 'E2', which uses it as a substrate. Thus in such a short pathway, the complex E1.I is a better substrate for E2, compared to the freely diffusible small molecule 'I'. Both the following reactions may occur and compete:
  • Direct transfer (channeling): ... E1.I + E2 --> E1.I.E2 --> E1 + E2.I --> ...
  • Release/rebinding ('normal'): ... E1.I --> E1+ I   and then    E2 + I --> E2.I --> ...
The occurence of channeling or direct transfer through transient association of enzymes during activity has important kinetic as well as regulatory implications. Indeed, in contrast with the 'normal mechanism', the metabolite 'I' is forced to go on through E2 rather than being available as a substrate to another enzyme 'En' in a different (competing?) pathway.
An extreme case for channeling is the permanent organization of enzymes in a pathway into a 'multi-enzyme complex', in which the intermediates are restricted into a common internal active locus. The complex represents a black box into which a substrate enters, is sequentially processed in isolation from the medium, and only the final product comes out and become available to further catalysis by other systems. A famous example for that is the pyruvate dehydrogenase complex of bacteria and mitochondria.

Physical process in which molecules move in space in the direction of their local concentration gradient. In cellular systems, this results in their net translocation from the site of their production to that of their utilization. Diffusion in aqueous solution is slow and may represent a rate limiting factor in heterogeneous catalytic systems operating at high rates of catalysis.

glucose 6 phosphate
The product of initial activation of glucose by ATP catalyzed by hexokinase. This results in trapping glucose inside cells, allowing it to proceed in glycolysis or gluconeogenesis. This central metabolite represents also an important modulator of enzymes/pathways in carbohydrate and energy metabolism. Specifically, it modulates both the activity and the localization of brain hexokinase.

This term qualifies systems or modes of catalysis often encountered in cellular biology, in which the functional components are not homogeneously distributed distributed as in solution, but rather organized.
Model systems have been described using immobilized enzymes at the surface of matrices, or within porous lattices, surrounded by bulk solution in which reactants are dissolved. In such heterogeneous catalytic systems, the substrates and products have to diffuse to and from the enzymic phase in order to complete the catalytic cycle as sensed by an external observer (e.g., in bulk solution). The effective properties of such a heterogeneous system displays a channeling behavior for the reactants and may strikingly differ from these of the same system in solution. They include in addition to intrinsic properties of the catalyst(s), other features inherent to the non-homogeneous character of the system.
The use of localized probes to monitor the concentration of reactants in the immediate vicinity of the catalyst(s) helps to resolve the contribution of physical processes (diffusion, chemical or electrostatic partition, local changes in pH, etc.) to the observed behavior of heterogeneous systems.

The first enzyme of glycolysis, converting intracellular glucose to Glucose-6-Phosphate, using the gamma phosphate of ATP. The enzyme in bacteria and yeast contains a single domain (50 kDa) and is located in the cytosol. In higher organisms, the enzyme is composed of two distinct domains similar in structure to the former, but differing in function:

A bioluminescent enzyme isolated from light organs of fireflies. It catalyzes light emission (yellow-green, 560 nm) from luciferin after its activation by ATP and oxidative decarboxylation (by molecular oxygen). The quantum yield for this reaction is extremely high (0.88 photon/cycle). Under proper experimental conditions, the light output is proportional to the concentration of the limiting substrate (e.g., ATP). This makes the enzyme an exquisitely specific and sensitive (pM range) probe for detection and monitoring of ATP.
Normally located in peroxisomes, this 550 residues (62 kDa) protein can be redirected to various cellular locations by genetic engineering, and may serve as a probe for protein traffic and assembly, or as a powerful localized probe for local [ATP] in studies of organized metabolism to detect diffusion effects.

Macromolecular crowding
In contrast to the dilute solutions in which classical enzymology studies are conducted, the interior of cells represents a very dense and crowded environment. Although the concentration of each defined molecular species is usually low (sub micromolar range), the combined density of "background" macromolecular components can reach very high values (200-300 mg protein/ml in the cytoplasm!).
In addition, cells contain a profusion of surfaces, like membranes and cytoskeletal fibres, which not only occupy a significant fraction of the cellular volume, but also provide a substrate upon which non-specific adsorption occurs. This leaves a very limited space to water, the major solvent, which gets organized into a few solvation layers, in contrast to the more familiar "bulk water" in dilute solution. In extreme cases, like in the matrix of mitochondria, macromolecules are said to exist and function in a quasi-crystalline state. This situation may represent a prerequisite for the self-organization characteristic of all living systems; thermodynamically, the process is entropy-driven due to water exclusion, similarly to protein folding.
A crowded physico-chemical environment bears several fundamental implications for the structure, function and evolution of cellular systems, and their experimental study:
Finally, extreme caution should be exerted in the extrapolation of experimental results (acquired in dilute solutions) to function in the intact cell.

Macromolecular recognition
In view of the inherently crowded nature of the intracellular milieu, most macromolecules are likely to be in physical contact with each other. Since all the components in a cell have evolved in parallel, they had ample opportunity to adapt to such environment (to the advantage of the organism) through molecular evolution. It is believed that confined macromolecules in each cell have reached mutual recognition for a better organization and coordinated function.
Cell biologists increasingly realize the importance of molecular recognition, and this topics is currently a favored subject of multidisciplinary research.
Macromolecular recognition (as protein folding) is determined by the sequence, 3D structure and the environment. It is effected by building blocks occuring at the surface of macromolecules, being complementary in terms of:
  • geometric fit: match at the surface needed to achieve maximal packing density;
  • hydrophobic fit: surface patches matching needed for water exclusion;
  • hydrogen bonding/electrostatic fit: for further stabilization of the associated molecules.
Most these factors are predictable through computer analysis of known 3D structures or existing models of macromolecules suspected to interact and the simulation of their mutual docking. Other (wet) approaches to detect macromolecular interaction are non-denaturating separation methods (ultracentrifugation, gel exclusion or electrophoresis), or recombinant DNA methods (e.g., yeast double hybrid system).

porin or VDAC
Mitochondrial outer membrane protein, also referred to as VDAC (Voltage Dependent Anion Channel). Represents a regulated gate between the cytosol and intramitochondrial compartments for the exchange of metabolites. Believed to occur as a dimer, this mostly beta sheet transmembrane protein forms a hydrophilic pore in the outer membrane allowing neutral molecules (up to 5 kDa) to diffuse in and out the mitochondrion.

outer membrane
The mitochondrial envelope separating internal compartments from the cytosol. Contact sites (with the inner membrane) have been described and proposed to harbor in addition to the mitochondrial protein import machinery, multi-enzyme complexes in which metabolites are channeled between the matrix and the cytosol. For example, the coupling factor (matrix), adenytate translocase (inner membrane), creatine kinase (inter membrane space), porin (outer membrane), and hexokinase or glycerol kinase (cytosol) are believed to form such a superstructure in contact sites.

organized metabolism
A new view of cellular metabolism starts to emerge, emphasizing the contribution of organization of catalytic structures upon intracellular surfaces (membranes and cytoskeleton), and the differences between heterogeneous and homogeneous (as in ideal solution) catalytic behavior. Thus, the dynamic association and/or segregation of enzymes (even in the cytosol) is believed to mediate channeling of reactants in the desired direction and/or pathway, according to metabolic needs. This property of enzyme to be dually located inside the cell is often referred to as 'ambiquity'.
The mutual affinity of protein pairs is encoded in their respective sequences and structures, while their potential association or separation is effected through simple physico-chemical determinants of their local environment. The latter are believed to be achieved through the 'interpretation' of variable signals in the global environment (extra- or intra-cellular) by proper signal transduction.

targeting or leader or signal sequence
A short sequence on a newly translated polypeptide which serves as a signal for cellular protein sorting and traffic machineries. This signal is recognized by compartment-specific receptors and presented to other components whose role is to transfer the polypeptide to the correct location, and assist in their post-translational processing and assembly.

Adenylate translocase or ATP/ADP exchanger is an integral mitochondrial inner membrane protein catalysing a fast 1:1 electrogenic exchange of ATP and ADP in their anionic form according to their respective electrochemical potential gradients across the membrane. The adenylate transport is specifically inhibited by atractylosides.

Last update: Apr 2003- Claude Aflalo
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