Enzyme dynamics during catalysis

Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory.     

How do enzymes work?

The principle of transition-state stabilization asserts that the occurrence of enzymic catalysis is equivalent to saying that an enzyme binds the transition state much more strongly than it binds the ground-state reactants. An outline of the origin and gradual acceptance of this idea is presented, and elementary transition-state theory is reviewed. It is pointed out that a misconception about the theory has led to oversimplification of the accepted expression relating catalysis and binding, and an amended expression is given. Some implications of the transition-state binding principle are then explored. The amended expression suggests that internal molecular dynamics may also play a role in enzymic catalysis. Although such effects probably do not make a major contribution, their magnitude is completely unknown. Two examples of recent advances due to application of the transition-state binding principle are reviewed, one pertaining to the zinc protease mechanism and the other to the generation of catalytic antibodies.     

Chemical aspects of enzyme inhibition

The use of chemical reagents as enzyme inhibitors has yielded information concerning the mechanism of inhibition and the role in catalysis played by active groups on the protein apoenzyme, the coenzyme, or the metal component. Inhibition analysis has also furnished valuable clues concerning the architecture, chemical properties, and catalytic mechanism of the active site of the enzyme. In many cases, in vivo effects of inhibitors can be closely correlated with in vitro inhibition of purified enzyme systems. The effects of antimicrobial and anticancer agents, insecticides, and drugs can often be explained in terms of enzyme inhibition. The design and synthesis of new inhibitors offers great promise when applied to the control of undesirable organisms and to the prevention and cure of disease in the immediate future.     

Modification of the active site of alkaline phosphatase by site-directed mutagenesis

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.     

A perspective on enzyme catalysis

The seminal hypotheses proposed over the years for enzymatic catalysis are scrutinized. The historical record is explored from both biochemical and theoretical perspectives. Particular attention is given to the impact of molecular motions within the protein on the enzyme's catalytic properties. A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion. Such coupled networks have important implications for the origin and evolution of enzymes, as well as for protein engineering.     

How enzymes work: analysis by modern rate theory and computer simulations

Advances in transition state theory and computer simulations are providing new insights into the sources of enzyme catalysis. Both lowering of the activation free energy and changes in the generalized transmission coefficient (recrossing of the transition state, tunneling, and nonequilibrium contributions) can play a role. A framework for understanding these effects is presented, and the contributions of the different factors, as illustrated by specific enzymes, are identified and quantified by computer simulations. The resulting understanding of enzyme catalysis is used to comment on alternative proposals of how enzymes work.     

The role of pair dispersion in turbulent flow

Mixing and transport in turbulent flows-which have strong local concentration fluctuations-are essential in many natural and industrial systems including reactions in chemical mixers, combustion in engines and burners, droplet formation in warm clouds, and biological odor detection and chemotaxis. Local concentration fluctuations, in turn, are intimately tied to the problem of the separation of pairs of fluid elements. We have measured this separation rate in an intensely turbulent laboratory flow and have found, in quantitative agreement with the seminal predictions of Batchelor, that the initial separation of the pair plays an important role in the subsequent spreading of the fluid elements. These results have surprising consequences for the decay of concentration fluctuations and have applications to biological and chemical systems.     

Enzymatic catalysis and transition-state theory

The application of transition-state theory to enzymatic catalysis provides an approach to understanding enzymatic catalysis in terms of the factors that determine the strength of binding of ligands to proteins. The prediction that the transition state should bind to the enzyme much more tightly than the substrate is supported by the experimental results with stable analogs of transition states. Transition-state analogs have great potential for use in understanding enzymatic catalysis and in inhibiting enzymes. Because of their potency and specificity as enzyme inhibitors, some of them may become very useful chemotherapeutic agents.     

The spatial dimension in population fluctuations

Theoretical research into the dynamics of coupled populations has suggested a rich ensemble of spatial structures that are created and maintained either by external disturbances or self-reinforcing interactions among the populations. Long-term data of the Canadian lynx from eight Canadian provinces display large-scale spatial synchrony in population fluctuations. The synchronous dynamics are not time-invariant, however, as pairs of populations that are initially in step may drift out of phase and back into phase. These observations are in agreement with predictions of a spatially-linked population model and support contemporary population ecology theory.     

Side-on copper-nitrosyl coordination by nitrite reductase

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.     

Chemistry. The motions of an enzyme soloist

Dynamics of proteins are crucial to their function. In his Perspective, Orrit stresses the advantages of studying these dynamics with single-molecule methods--which require no synchronization--rather than with conventional ensemble measurements. He highlights the report by Yang et al., who follow the fluorescence of a single enzyme molecule. Electron transfer from the fluorophore to a quencher induces fluctuations of the fluorescence lifetime along with the fluorophore-quencher distance. The wide range of characteristic times of those fluctuations reveals the complexity of the protein's potential energy landscape. As a new molecular ruler, electron transfer complements other single-molecule methods such as energy transfer (FRET) for distances shorter than a few nanometers.     

The chemistry of self-splicing RNA and RNA enzymes

Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.     

分子印迹技术及其应用研究进展

分子印迹技术是20世纪90年代迅速发展起来的一种化学分析技术,属于超分子化学研究范畴,通常被人们描述为制造识别“分子钥匙”的人工“锁”技术。它在化学仿生传感器、模拟抗体、模拟酶催化、膜分离技术、色谱中对映体和位置异构体的分离、固相提取、临床药物分析等领域展现了良好的应用前景。本文就分子印迹技术的产生、分析原理和应用研究进展进行综述。     

反胶束体系中的酶催化反应

反胶束是新的酶催化反应的介质工程,酶在反胶束体系中的性质与在水溶液中相比有较大区别。综述了含酶反胶束体系的制备、反胶束体系中影响酶催化化学反应的因素,以及在反胶束体系中酶的活性及动力学特性,介绍了反胶束体系下酶催化反应的优点及应用,并展望了其发展前景。     

Tinkering with enzymes: what are we learning?

It is now possible, by site-directed mutagenesis of the gene, to change any amino acid residue in a protein to any other. In enzymology, application of this technique is leading to exciting new insights both into the mechanism of catalysis by particular enzymes, and into the basis of catalysis itself. The precise and often delicate changes that are being made in and near the active sites of enzymes are illuminating the interdependent roles of catalytic groups, and are allowing the first steps to be taken toward the rational alteration of enzyme specificity and reactivity.     

Biogeochemistry. Sulfate reducers--dominant players in a low-oxygen world?

Sulfate reducing bacteria can adapt to extreme physical and chemical conditions and play an important role in global geochemical cycles, but their role in the formation of ore deposits has remained controversial. Strong support for such a role is provided by Labrenz et al., who have discovered sulfate-reducing bacteria that can tolerate low levels of oxygen and can precipitate zinc sulfide minerals. The results may have implications for bioremediation and may provide clues to processes that may have been more widespread in the geologic past.     

Studying enzyme mechanism by 13C nuclear magnetic resonance

High-resolution carbon-13 nuclear magnetic resonance (NMR) spectra of enzyme-inhibitor and enzyme-substrate complexes provide detailed structural and stereochemical information on the mechanism of enzyme action. The proteases trypsin and papain are shown to form tetrahedrally coordinated complexes and acyl derivatives with a variety of compounds artificially enriched at the site or sites of interest. These results are compared with the structural information derived from x-ray diffraction. Detailed NMR studies have provided a clearer picture of the ionization state of the residues participating in enzyme-catalyzed processes than other more classical techniques. The dynamics of enzymic catalysis can be observed at sub-zero temperatures by a combination of cryoenzymology and carbon-13 NMR spectroscopy. With these powerful techniques, transient, covalently bound intermediates in enzyme-catalyzed reactions can be detected and their structures rigorously assigned.     

Electron localization determines defect formation on ceria substrates

The high performance of ceria (CeO2) as an oxygen buffer and active support for noble metals in catalysis relies on an efficient supply of lattice oxygen at reaction sites governed by oxygen vacancy formation. We used high-resolution scanning tunneling microscopy and density functional calculations to unravel the local structure of surface and subsurface oxygen vacancies on the (111) surface. Electrons left behind by released oxygen localize on cerium ions. Clusters of more than two vacancies exclusively expose these reduced cerium ions, primarily by including subsurface vacancies, which therefore play a crucial role in the process of vacancy cluster formation. These results have implications for our understanding of oxidation processes on reducible rare-earth oxides.     

X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor

Nitrogenase is a complex enzyme that catalyzes the reduction of dinitrogen to ammonia. Despite insight from structural and biochemical studies, its structure and mechanism await full characterization. An iron-molybdenum cofactor (FeMoco) is thought to be the site of dinitrogen reduction, but the identity of a central atom in this cofactor remains unknown. Fe Kβ x-ray emission spectroscopy (XES) of intact nitrogenase MoFe protein, isolated FeMoco, and the FeMoco-deficient nifB protein indicates that among the candidate atoms oxygen, nitrogen, and carbon, it is carbon that best fits the XES data. The experimental XES is supported by computational efforts, which show that oxidation and spin states do not affect the assignment of the central atom to C(4-). Identification of the central atom will drive further studies on its role in catalysis.     

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.