Transport catalysis

Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This “induced transition fit” (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for “low affinity” transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.     

Transport catalysis

Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This "induced transition fit" (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for "low affinity" transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.     

RNA catalysis

Our understanding of the relationship between the structure of RNA and its catalytic activity has advanced significantly in the past year. These advances include time-resolved crystallographic studies on the hammerhead ribozyme, as well as new structures of a group I intron, a lead(II)-cleavage ribozyme, a hepatitis delta virus ribozyme, and components of the spliceosome machinery and the peptidyl transferase center of the ribosome and, most significantly, the structure of the ribosome itself.     

RNA folding and catalysis

Catalysis in RNA is intimately connected to the folding. The small nucleolytic ribozymes function by a nucleophilic attack of the 2-oxygen on the 3-phosphate, in an S_N2 mechanism. This requires an alignment of the 2-O, 3-P and 5-O, that does not occur in normal A-form RNA. It is therefore likely that structural distortion plays a major role in the enhancement of the reaction rate, facilitating the trajectory into the in-line transition state. Given the polyelectrolyte nature of nucleic acids, metal ions are critical to folding processes in RNA. We have shown that two small nucleolytic ribozymes, the hammerhead and hairpin ribozymes, undergo metal ion-induced folding processes. The hammerhead ribozyme folds in two stages, each of which is induced by the binding of a single structural ion. The first corresponds to the formation of the ribozyme scaffold, while the second is the formation of the catalytic core of the ribozyme. By contrast, the hairpin ribozyme undergoes a single folding event induced by the binding of at least two metal ions, and involves the close interaction between two internal loops to form the active ribozyme.This revised version was published online in October 2005 with corrections to the Cover Date.     

Interfacial catalysis by lipases

We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8).

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.     

Allostery in enzyme catalysis

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Mechanisms of RNA catalysis

Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.     

Studies of translocation catalysis

There is a symbiotic relationship between the evolution of fundamental theory and the winning of experimentally-based knowledge. The impact of the General Chemiosmotic Theory on our understanding of the nature of membrane transport processes is described and discussed. The history of experimental studies on transport catalysed by ionophore antibiotics and the membrane proteins of mitochondria and bacteria are used to illustrate the evolution of knowledge and theory. Recent experimental approaches to understanding the lactose-H⁺ symport protein ofEscherichia coli and other sugar porters are described to show that the lack of experimental knowledge of the three-dimensional structures of the proteins currently limits the development of theories about their molecular mechanism of translocation catalysis.     

Toward bifunctional antibody catalysis

Antibodies that catalyze the deprotonation of unactivated benzisoxazoles to give the corresponding salicylonitriles were prepared using as antigen a 2-aminobenzimidazolium derivative coupled to a carrier protein via its benzene ring. The hapten was designed to induce an antibody binding site with both a base and an acid, in position to initiate proton transfer and stabilize developing negative charge at the phenoxide leaving group, respectively. Consistent with this design, the catalysts exhibit bell-shaped pH-rate profiles, while chemical modification identified several functional groups that could participate in bifunctional catalysis. One of the antibodies, 13G5, is particularly notable in catalyzing the elimination of 6-glutaramidebenzisoxazole with a > 10(5)-fold rate acceleration over background and an effective molarity of > 10(4) M for its catalytic base. These properties compare favorably to the efficiencies achieved by the best previously characterized antibodies with substantially more reactive substrates.     

Principles of antibody catalysis

Antibodies have now been shown to catalyze a variety of chemical transformations, including hydrolytic, concerted, and bimolecular reactions. The inherent chirality of the antibody binding pocket has been exploited to exert precise stereochemical control over their catalyzed reactions. The mechanisms by which antibodies catalyze reactions are not expected to differ in any general way from those of natural enzymes. Antibodies use their binding energy to stabilize species of higher free energy which appear along the reaction coordinate or effect general acid/base catalysis. The advent of catalytic antibodies promises new catalysts that extend the range of catalysis by proteins to chemical transformations that were not required during the evolution of enzymes.     

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).