Magnesium magnetic isotope effect: A key to the mechanochemistry of phosphorylating enzymes as molecular machines

The discovery of the powerful magnesium isotope effect on enzymatic ATP synthesis provides a new insight into the mechanochemistry of enzymes as molecular machines. The catalytic activities of ATPase, creatine kinase, and glycerophsphate kinase containing a Mg²⁺ ion with magnetic isotope nuclei (²⁵Mg) were found to be two to four times higher than those of the enzymes with spinless, nonmagnetic magnesium cation isotopes (²⁴Mg or ²⁶Mg). This demonstrates unambiguously that ATP synthesis is a spin-selective process involving Mg2+ as the electron-accepting reagent. ATP synthesis proceeds in an ion-radical pair consisting of an ADP oxyradical and Mg²⁺. In this process, the magnesium bivalent cation is the key agent that transforms the mechanics of a protein molecule into chemical processes. This ion is the crucial structural component of enzymes as mechanochemical molecular machines.     

Fluctuation as a tool of biological molecular machines

The mechanism for biological molecular machines is different from that of man-made ones. Recently single molecule measurements and other experiments have revealed unique operations where biological molecular machines exploit thermal fluctuation in response to small inputs of energy or signals to achieve their function. Understanding and applying this mechanism to engineering offers new artificial machine designs.     

C-Glucosylated malonitrile as a key intermediate towards carbohydrate-based glycogen phosphorylase inhibitors

Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxadiazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phosphorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range. In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydropyrimidin-2-one may bind at the catalytic site.     

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.     

Gap junctions: towards a molecular structure

Gap junctions are ubiquitous plasma membrane specializations that allow cells to exchange small molecules and ions directly. The isolation, biochemical characterization and molecular cloning of the major protein of rat liver gap junctions lead to a clearer view of these membrane zones that allow cells to ‘talk’ to each other and co-ordinate their activities in tissues and organs.     

Plaque imaging and characterization using magnetic resonance imaging: towards molecular assessment

Identification of high-risk atherosclerotic lesions prone to rupture and thrombosis may greatly decrease the morbidity and mortality associated with atherosclerosis. High-resolution magnetic resonance imaging (MRI) has recently emerged as one of the most promising techniques for the non-invasive study of atherothrombotic disease, as it can characterize plaque composition and monitor its progression. The development of MRI contrast agents that specifically target components of the atherosclerotic plaque may enable non-invasive detection of high-risk lesions. This review discusses the use of high-resolution MRI for plaque detection and characterization and the potentials of "Molecular Imaging" using a variety of molecules present in atherosclerotic plaques that may serve as targets for specific contrast agents to allow the identification of high-risk atherosclerotic lesions in-vivo. Ultimately, such agents may enable treatment of "high-risk" patients prior to lesion progression and occurrence of complications.     

The emergence of molecular machines as a prerequisite of the ancient RNA world evolution

The problem of the start of biological evolution in the ancient RNA world is considered. It is postulated that the appearance of catalytic RNAs — ribozymes — via spontaneous cis- and trans-rearrangements of polyribonucleotides in primordial Darwin ponds should not have been sufficient for the start of evolution, until a new class of functional RNA, namely energy-dependent molecular machines, arose. The proposed hypothesis is that the simplest and primary type of molecular machines could be nucleoside triphosphate-dependent RNA-based helicases, which were capable of unwinding the stable double-helical RNAs inevitably formed during RNA syntheses on complementary templates. Thereupon, unwinding RNA polymerases could appear as a result of association or fusion of helicases and polyribonucleotide-polymerizing ribozymes. The latter event provided the mechanism of RNA replication using the double-helical RNAs as a communal genofond (gene pool) of a Darwin pond, and thus initiated the fast evolution of the ancient RNA world.     

Computational studies of molecular machines: the ribosome

The past decade has produced an avalanche of experimental data on the structure and dynamics of the ribosome. Groundbreaking studies in structural biology and kinetics have placed important constraints on ribosome structural dynamics. However, a gulf remains between static structures and time dependent data. In particular, X-ray crystallography and cryo-EM studies produce static models of the ribosome in various states, but lack dynamic information. Single molecule studies produce information on the rates of transitions between these states but do not have high-resolution spatial information. Computational studies have aided in bridging this gap by providing atomic resolution simulations of structural fluctuations and transitions between configurations.     

Cell death by necrosis: towards a molecular definition

Necrosis has been defined as a type of cell death that lacks the features of apoptosis and autophagy, and is usually considered to be uncontrolled. Recent research suggests, however, that its occurrence and course might be tightly regulated. After signaling- or damage-induced lesions, necrosis can include signs of controlled processes such as mitochondrial dysfunction, enhanced generation of reactive oxygen species, ATP depletion, proteolysis by calpains and cathepsins, and early plasma membrane rupture. In addition, the inhibition of specific proteins involved in regulating apoptosis or autophagy can change the type of cell death to necrosis. Because necrosis is prominent in ischemia, trauma and possibly some forms of neurodegeneration, further biochemical comprehension and molecular definition of this process could have important clinical implications.     

Mitosis: towards a molecular understanding of chromosome behavior.

The past year has seen important contributions made to resolving how chromosomes attach to and move on the mitotic spindle of animal cells. These include the findings that: kinetochore microtubules are derived from the asters (i.e. centrosomes); poleward chromosome motion need not be coupled to kinetochore microtubules disassembly; the motor for poleward chromosome motion is associated with the kinetochore; and immunological evidence that this motor is cytoplasmic dynein.     

Reverse engineering a protein: the mechanochemistry of ATP synthase

ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers.     

The release of acetylcholine: from a cellular towards a molecular mechanism

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge. Consequently, the mechanism releasing ACh from the cytoplasm in a packet form was searched at the presynaptic membrane itself. With synaptosomes isolated from the electric organ of Torpedo, it became possible to freeze them rapidly at the peak of ACh release and study their membrane and contents after cryofracture. A statistical analysis showed that the main structural change was the occurrence of large intramembrane particles at the peak of ACh release and under all release conditions. This impressive change contrasted with the stability in the number of vesicles. Another role for the vesicle was envisaged during intense stimulations when the cytoplasmic ACh and ATP pools become exhausted. The decrease in ATP leads to an increase in calcium and protons in the cytoplasm; this signals the depletion of vesicular ACh and ATP stores in the cytoplasm. Release can go on, while ATP promotes the uptake of calcium by vesicles. At the end of its cycle the vesicle will be full of calcium and will perhaps release it. As far as the mechanism of ACh release is concerned it probably depends on a membrane component (perhaps the large particles) activated by calcium and able to translocate ACh in a quantal or subquantal form. In most recent work we showed that if a lyophilized presynaptic membrane was used to make proteoliposomes filled with ACh, they released ACh upon calcium action.