Protein dynamics and enzyme catalysis: the ghost in the machine?

One of the most controversial questions in enzymology today is whether protein dynamics are significant in enzyme catalysis. A particular issue in these debates is the unusual temperature-dependence of some kinetic isotope effects for enzyme-catalysed reactions. In the present paper, we review our recent model [Glowacki, Harvey and Mulholland (2012) Nat. Chem. 4, 169-176] that is capable of reproducing intriguing temperature-dependences of enzyme reactions involving significant quantum tunnelling. This model relies on treating multiple conformations of the enzyme-substrate complex. The results show that direct 'driving' motions of proteins are not necessary to explain experimental observations, and show that enzyme reactivity can be understood and accounted for in the framework of transition state theory.     

Enthalpy–entropy compensation and the isokinetic temperature in enzyme catalysis

Enthalpy–entropy compensation supposes that differences in activation enthalpy ?H ^? for different reactions (or, typically in biochemistry, the same reaction catalysed by enzymes obtained from different species) may be compensated for by differences in activation entropy ?S ^?. At the isokinetic temperature the compensation is exact, so that all samples have the same activity. These ideas have been controversial for several decades, but examples are still frequently reported as evidence of a real phenomenon, nearly all of the reports ignoring or discounting the possibility of a statistical artefact. Even for measurements in pure chemistry artefacts occur often, and they are almost inescapable in enzyme kinetics and other fields that involve biological macromolecules, on account of limited stability and the fact that kinetic equations are normally valid only over a restricted range of temperature. Here I review the current status and correct an error in a recent book chapter.     

Computational modeling of substrate specificity and catalysis of the β-secretase (BACE1) enzyme

In this combined MD simulation and DFT study, interactions of the wild-type (WT) amyloid precursor protein (APP) and its Swedish variant (SW), Lys670 → Asn and Met671 → Leu, with the beta-secretase (BACE1) enzyme and their cleavage mechanisms have been investigated. BACE1 catalyzes the rate-limiting step in the generation of 40-42 amino acid long Alzheimer amyloid beta (Aβ) peptides. All key structural parameters such as position of the flap, volume of the active site, electrostatic binding energy, structures, and positions of the inserts A, D, and F and 10s loop obtained from the MD simulations show that, in comparison to the WT-substrate, BACE1 exhibits greater affinity for the SW-substrate and orients it in a more reactive conformation. The enzyme-substrate models derived from the MD simulations were further utilized to investigate the general acid/base mechanism used by BACE1 to hydrolytically cleave these substrates. This mechanism proceeds through the following two steps: (1) formation of the gem-diol intermediate and (2) cleavage of the peptide bond. For the WT-substrate, the overall barrier of 22.4 kcal/mol for formation of the gem-diol intermediate is 3.3 kcal/mol higher than for the SW-substrate (19.1 kcal/mol). This process is found to be the rate-limiting in the entire mechanism. The computed barrier is in agreement with the measured barrier of ca. 18.00 kcal/mol for the WT-substrate and supports the experimental observation that the cleavage of the SW-substrate is 60 times more efficient than the WT-substrate.     

Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis — possible states and configurations of manganese and water

Absorption changes coupled with the individual transitions S₀–S₃ and redox reactions in the water-splitting enzyme system S of photosynthesis have been measured. The principal difficulties of measuring the very small absorption changes in the ultraviolet coupled with those reactions have been reduced drastically through the use of a highly purified Photosystem II complex isolated from the Cyanobacterium synechococcus. The general problem caused by the mixing of the S states during a train of flashes and the falsification through the overlap with absorption changes of Q_B (binary oscillations) have been treated as follows. (1) The binary oscillations were bypassed through the use of silicomolybdate and high concentrations of DCBQ, respectively, as external electron acceptor. (2) Stable absorption changes of the mixed S-state transitions have been deconvoluted through fitting procedures to get the changes of the individual transitions of S₁ → S₂ → S₃ → S₀ → S₁. (3) Kinetically resolved absorption changes of the S-states in the 100-μs range gave independent information on the individual transitions. (4) Stable absorption changes of the S₀ → S₁ transitions in the forefront were induced after shifting the S states through low concentrations of NH₂OH two units backwards. Analysis of the resulting sequence S_x → S₀ → S₁ → S₂ → S₃ → S₀, beginning with an NH₂OH depending pre-state, S_x, and followed by an S₀ → S₁ transition not mixed with the opposite S₃ → S₀ transition, increased the conclusiveness considerably. It results that the ultraviolet spectrum of the S₀ → S₁ transition is different from the spectra of the S₁ → S₂ and S₂ → S₃ transition. Possible states of manganese, water and surplus charges responsible for these spectra are presented.     

Does cypermethrin affect enzyme activity,respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

Insecticides cause a range of sub‐lethal effects on targeted insects, which are frequently detrimental to them. However, targeted insects are able to cope with insecticides within sub‐lethal ranges, which vary with their susceptibility. Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) to sub‐lethal exposure to the pyrethoid insecticide cypermethrin. We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure. Lower respiration rate and lower activity were also expected in insecticide‐resistant insects since these traits are also likely to favor survivorship under insecticide exposure. Curiously though, cypermethrin did not affect activity of digestive and energy metabolism enzymes, and even reduced the activity of some enzymes (particularly for cellulase and cysteine‐proteinase activity in this case). There was strain variation in response, which may be (partially) related to insecticide resistance in some strains. Sub‐lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects, which is likely to impair their fitness. However, such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced). Walking activity varies with strain and may minimize insecticide exposure, which should be a concern, particularly if associated with (physiological) insecticide resistance.     

Tissue distribution of the “N-end rule” ubiquitin-conjugating enzyme, HR6, in the rat

The conjugation of multiple ubiquitin molecules is required for recognition and degradation of a protein by the proteasome. The ubiquitination pathway responsible for the bulk of constitutive protein degradation targets proteins carrying basic or large hydrophobic amino acids at the N-terminus. In mammalian cells, this N-end rule pathway requires the ubiquitin-conjugating enzyme HR6. Until now, it has not been known which mammalian tissues and cell types predominantly utilize this pathway for degradation. Therefore, the distribution and intracellular localization of HR6 was determined by indirect immunofluorescence techniques and protein blotting of adult rat tissues. Intense immunoreactivity against HR6 was detected in various epithelia, muscle, testis, peripheral neurons, chromaffin cells and macrophages, whereas lower HR6 protein levels were found in the gut or in the kidney. Autonomic and sensory neurons, glandular cells and spermatocytes revealed prominent nuclear HR6 immunoreactivity. Plasma membrane labeling was observed in peripheral neurons, spermatocytes and skeletal muscle cells. Smooth muscle cells, macrophages, endothelial and epithelial cells exhibited primarily cytoplasmic staining. The clear differences in the regional and intracellular distribution of HR6 are suggestive for the involvement of N-end rule protein degradation in various physiological processes dependent on cell type and subcellular structure.     

Folding dynamics of phenylalanine hydroxylase depends on the enzyme’s metallation state: the native metal,iron, protects against aggregate intermediates

Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for the phenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU). To better understand how protein structure and folding profiles are affected by the metal cofactor, we investigated the chemical (un)folding of apo- and holo-PAH from Chromobacterium violaceum (cPAH) using circular dichroism (CD) and analytical ultracentrifugation (AUC). Holo-cPAH shows a two-state unfolding transition. In contrast, the unfolding profile for apo-cPAH reveals a three-state (un)folding pathway and accumulation of an intermediate (apo-cPAH^I). This intermediate is also observed in refolding experiments. Fluorescence studies are consistent with the CD findings. The intermediate apo-cPAH^I and unfolded state(s) of apo- and holo-cPAH^U have been characterized by analytical ultracentrifugation (AUC). At 2.4 and 2.8 M GuHCl, 90% of the signal for apo-cPAH has a weight average sedimentation coefficient in water at 20°C (s20,w) of about 48 S, representing multiple aggregate species made of multiple monomers of cPAH. Aggregate formation for apo-cPAH is also confirmed by dynamic light scattering and electron microscopy giving a hydrodynamic radius (R_H) of 41 nm for apo-cPAH^I versus 3.5 nm for the native protein.     

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

Fractality, “coastline of the universe”, movement of the Earth, and “macroscopic fluctuations”

The evolution of views on the nature of the phenomenon of “macroscopic fluctuations”, discovered about sixty years ago as “anomalous scattering of results” of measuring the actomyosin enzyme activity, is traced in the paper. Since then, the general character of this phenomenon was stated because it was found in measurements of processes of different nature, being caused by movement of the Earth in heterogeneous and anisotropic space-time. The paper is dedicated to the memory of L.A. Blumenfeld: a many-decade discussion with him supported these investigations.     

Rarity,ecological memory,rate of floral change in phytoplankton—and the mystery of the Red Cock

In this article, we attempt to estimate the contemporary phytoplankton species pool of a particular lake, by assessing the rate of floral change over a period of 15 years. Phytoplankton time series data from Lake Stechlin, an oligo-mesotrophic lake in the Baltic Lake District (Germany) were used. Of the 254 algal species recorded during the 15-year of studies with roughly biweekly sampling, 212 species were planktonic. In the individual plankton years, the recorded total number of species changed between 97 and 122, of which the number of dominants (>1% contribution to the annual average of total biomass) was only 10–19. The 15-year cumulative number of species exhibited an almost linear increase after an initial saturation phase. This increase was attributed to two reasons: increase of sample size and immigration of species new to the flora. Based on a probabilistic model developed in this study, we estimated the number of co-existing planktonic species of the lake as some 180, and the rate of floral change as 1–2 species per year. Of these co-existing species, only few maintain the matter–energy processing ecosystem functions in any particular plankton year. Selection of these dominants is probably driven by mesoclimatic cycles, coupled with human-induced forcing, like eutrophication. All others are hiding as an ecological memory, in the sense of the capacity or experiences of past states to influence present or future responses of the community. Data analyses suggest that selection of the ‘memory species’ that show temporary abundance increases over shorter (several years) periods are largely dependent upon the dominants. These results show that interspecific interactions and the particular autecological features of the dominants, together with their effects on the whole ecosystem, act as a major organizing force. Some phytoplankton species, like Planktothrix rubescens, are efficient ecosystem engineers with cascading effects of both a top-down and bottom-up nature. Historical scientific data on Planktothrix blooms in Lake Stechlin suggest cyclic patterns in long-term development of phytoplankton which, as the legend of the Red Cock suggests, dates back much further than scientific archives.     

Comparative Study of the Absorption,Plasma Levels,and Urinary Excretion of the “New” and the “Old” Lanoxin

A comparative study was performed of the absorption, the plasma level at equilibrium, and the urinary excretion of digoxin using two types of Lanoxin tablets, those produced before and after the 1972 alteration of the tablet manufacture.After a single dose the absorption rate of the new tablets was about twice as great as the old, both in young subjects and in the elderly patients. There were no significant differences in the plasma levels of digoxin for the two tablets 15 hours after the last administration in patients on an equal maintenance dose. The urinary excretion of digoxin increased about 40% when the “old” Lanoxin was replaced by the “new.” In elderly patients a daily dose of 0·125 mg twice daily of the new tablets should be sufficient to reach the therapeutic range. Young people need a higher dosage. If the kidney function is reduced by as much as 50% the dose should be reduced.     

Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?

Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels.     

Developmental dissociation of myelin synthesis and “myelin-associated” enzyme activities in the shiverer mouse

Developmental changes in three enzymes associated with myelin lipids were studied in the shiverer mouse, a murine mutant showing a severe deficiency of CNS myelin. Age-related changes in cerebroside sulfotransferase (measured in brain) and arylsulfatase A and cerebroside B-galactosidase (measured in brain and liver) were the same for shiverer and control mice. The shiverer mouse, therefore, demonstrates a dissociation between the genetic mechanisms regulating myelination in the CNS and developmental changes in enzyme activities thought to be closely related to the synthesis of myelin. In addition, we found no defect in the shiverer mouse in the incorporation of glycine-labeled basic protein into CNS myelin, indicating an important metabolic difference between the morphologically similar shiverer and quaking mutants.     

Rôle of the midgut,crop, and maxillae of Bombyx mori in the production of cocoon-digesting enzyme

The rôle of the midgut, crop, and maxillae in the production and utilization of the cocoon-digesting enzyme was investigated in the silkworm, Bombyx mori.About a sixtyfold purified preparation of midgut protease was obtained by ammonium sulphate precipitation and column chromatography.Immunological studies by the agar diffusion method of Ouchterlony revealed that the crop and midgut proteases of the pharate adult are antigenically identical whereas that of the maxillary protease is different.From the results of extirpation experiments and previous studies it was shown that the midgut, crop, and maxillae play important rôles in the escape of moths from their cocoons.     

Interactions of five- and six-membered cyclic esters with α-chymotrypsin: Kinetic evidence for covalent enzyme intermediates

Interactions of α-chymotrypsin with 2-coumaranone (I), 3,4-dihydrocoumarin (II), o-hydroxy-α-toluenesulfonic acid sultone (III), and β-o-hydroxyphenylethanesulfonic acid sultone (IV) were studied in the presence of 14% acetonitrile at pH 7.0 by means of the proflavin displacement technique and by inhibition of N-acetyl-l-tryptophan ethyl ester (ATrEE) hydrolysis. Under saturating conditions of either I, II, or III, an enzyme intermediate was shown to accumulate using either the proflavin displacement technique or the ATrEE activity assay. The intermediates have characteristics of covalent enzyme-substrate compounds and are believed to decompose simultaneously by two pathways, one to give free enzyme and hydrolyzed cyclic ester, and the other to give the original cyclic ester and free enzyme. With α-chymotrypsin and III the observed first-order rate constant for decomposition of the intermediate by the two pathways was 0.19 ± 0.04 min^?1, while the rate constant for the hydrolytic pathway alone was 0.013 ± 0.0009 min^?1. These results indicate that the covalent-like intermediate with this sultone is not only capable of reverting to starting cyclic ester but prefers this pathway over hydrolysis. Sultone IV was found to bind to enzyme; but in contrast to the behavior of esters I–III, the binding did not result in accumulation of a covalent-like intermediate.     

Oncosuppressive and oncogenic activity of the sphingolipid-metabolizing enzyme β-galactosylceramidase

β-galactosylceramidase (GALC) is a lysosomal enzyme that removes β-galactose from β-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression.