Protein dynamics and enzyme catalysis: insights from simulations

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Protein folding cooperativity: basic insights from minimalist models

Basic concepts about two-state, cooperative protein folding and its relation to first-order phase transitions are reviewed. Minimalist models capable of reproducing the required free energy barrier between folded and unfolded macroscopic states are described. A significantly more restrictive "calorimetric" criterion is also discussed, which is based on direct comparison between model and experimental heat capacities with additional assumptions about conformational enthalpy variation in the unfolded state.     

Age-related effects in enzyme catalysis

Summary Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue.Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in old glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of young glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native old glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.Abbreviations CPL circular polarization of luminescence - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - GPDH D-glyceraldehyde-3-phosphate dehydrogenase - ENAD⁺ nicotinamide 1,N⁶-ethenoadenine dinucleotide     

The effects of iron upon the radiation damage in non-heme iron proteins

Summary Radical yield measurements on irradiated ferritin and transferrin in the dry state have been carried out in order to test the role played by iron ions present in protein molecules on the radioresistance of these substances. In both cases, despite the high quantity of iron contained in ferritin and the direct linkage of two iron ions to the transferrin molecule, no difference between the radiation effects in native and iron-free proteins was observed.In the case of transferrin the lack of induction of secondary sulfur radicals by the presence of iron was observed.Some hypotheses about the interpretation of experimental results are discussed.This work was supported by C.N.R., Italy (grant no. 69.00682).We would like to express our thanks to the Paramagnetic Resonance Group of the University of Parma for permission and assistance in the use of EPR apparatus.     

Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, approximately 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination.     

A hemoprotein from azotobacter containing non-heme iron: isolation and crystallization

A new type of hemoprotein was isolated from extracts of $\text{Azotobacter vinelandii}$ using a heat step, application of the protamine-cellulose phosphate procedure, and precipitation with MgCl₂. Crystallization was achieved using low concentration of MgCl₂. The protein was shown to be a unique cytochrome of the b type containing up to 20% non-heme iron but no “acid labile sulfur”. Both the heme and non-heme iron appear to be functional redox components.     

Mechanistic insights from reversible splicing catalysis

Recent work demonstrating the ability of spliceosomes purified after the second catalytic step of splicing to efficiently reverse both steps of the reaction provides answers to several unresolved questions regarding the splicing reaction, and raises many more.     

Microanalysis of non-heme iron in animal tissues

The method recommended by the Iron Panel of the International Committee for the Standardization in Haematology for measurement of serum iron was adapted for measurement of non-heme iron in animal tissues. The method developed was designed specifically to facilitate measurement of non-heme iron using as little as 10 mg of tissue, in a final reaction volume of 60 microl. In this assay, tissue homogenates are treated with hydrochloric acid and trichloroacetic acid and heated at 95 degrees C. Non-heme iron is released and protein is precipitated. Following centrifugation, iron in the supernatant is reacted with ferrozine in the presence of the reducing agent thioglycolic acid, and the complex is quantified by spectrophotometry. The method was validated by analysis of two Standard Reference Materials (bovine liver), comparing results of this assay against certified values and concentrations determined by flame atomic absorption spectrometry following acid digestion. Results using this method for analysis of non-heme iron in guinea pig tissues (liver, kidney and heart) compared favorably with those obtained using micro-scale adaptations of three published reference methods. The new method was more sensitive, required less time, and was less cumbersome than the three published methods to which it was compared.     

Molecular insights of SAH enzyme catalysis and implication for inhibitor design

Biological transmethylation reaction is a key step in the duplication of virus life cycle, in which S-adenosylmethionine plays as the methyl donor. The product of this reactions, S-adenosylhomocysteine (AdoHcy) inhibits the transmethylation process. AdoHcy is hydrolysed to adenosine and L-homocysteine by the action of S-adenosylhomocysteine hydrolase (SAH). Thus the virus life cycle should be cut off once the action of SAH is inhibited. Our study was focussed on the discovery of potential inhibitor against SAH. We performed a similarity search in Traditional Chinese Medicine Database and retrieved 17 hits with high similarity. After that we virtually docked the 17 compounds as well as the natural substrates to the hydrolase using Autodock 3.0.1 software. Then we discussed about the mechanism of the inhibition reaction, followed by proposing the potential inhibitors by comparing best docked solutions and possible modification for the best inhibitors.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

In this minireview, we provide an account of the current state-of-the-art developments in the area of mono- and binuclear non-heme enzymes (NHFe and NHFe₂) and the smaller NHFe₍₂₎ synthetic models, mostly from a theoretical and computational perspective. The sheer complexity, and at the same time the beauty, of the NHFe₍₂₎ world represents a challenge for experimental as well as theoretical methods. We emphasize that the concerted progress on both theoretical and experimental side is a conditio sine qua non for future understanding, exploration and utilization of the NHFe₍₂₎ systems. After briefly discussing the current challenges and advances in the computational methodology, we review the recent spectroscopic and computational studies of NHFe₍₂₎ enzymatic and inorganic systems and highlight the correlations between various experimental data (spectroscopic, kinetic, thermodynamic, electrochemical) and computations. Throughout, we attempt to keep in mind the most fascinating and attractive phenomenon in the NHFe₍₂₎ chemistry, which is the fact that despite the strong oxidative power of many reactive intermediates, the NHFe₍₂₎ enzymes perform catalysis with high selectivity. We conclude with our personal viewpoint and hope that further developments in quantum chemistry and especially in the field of multireference wave function methods are needed to have a solid theoretical basis for the NHFe₍₂₎ studies, mostly by providing benchmarking and calibration of the computationally efficient and easy-to-use DFT methods.     

Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr‐I and Dsr‐II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α₂β₂γ₂ structure of Dsr‐I contains eight [4Fe–4S] clusters, two saddle‐shaped sirohaems and two flat sirohydrochlorins. In Dsr‐II, the [4Fe–4S] cluster associated with the sirohaem in Dsr‐I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr‐I and Dsr‐II confirm the co‐factor structures, whereas EPR of a third but inactive form, Dsr‐III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr‐I and Dsr‐II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ‐subunit C‐terminus is inserted into a positively charged channel formed between the α‐ and β‐subunits, with its conserved terminal Cysγ104 side‐chain covalently linked to the CHA atom of the sirohaem in Dsr‐I. In Dsr‐II, the thioether bond is broken, and the Cysγ104 side‐chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S₃O₆^2?, S₂O₃^2? and S^2?.     

Non-ferritin, non-heme iron pools in rat tissues

Concentrations of intracellular, low molecular weight (LMW) and desferrioxamine B (DF) chelatable Fe, in tissues of normal, Fe-deficient and Fe-loaded female rats, were determined. Ice cold, high speed supernatants were rapidly fractionated on Ultrogel AcA202 or by filter centrifugation. After correction for residual blood and DF effects on Fe proteins, liver, kidney, heart and spleen contained 3-8 micrograms/g LMW Fe, brain 20 micrograms/g, with DF; two-thirds of this was detected without DF. There was little variation with Fe status. MW standardization and fractionation on Sephadex G-25 indicated components of apparent MW 13,000, 1400 and 350; the latter two were rapidly labeled with in vivo 59Fe.     

Analysis of ground-state and transition-state effects in enzyme catalysis.

"The entire and sole source of catalytic power is the stabilization of the transition state; reactant-state interactions are by nature inhibitory and only waste catalytic power". So reads a literature quote expressing the current view on enzyme catalysis proposed by Pauling over 40 years ago. Its validity is now examined by means of a "split-site" model in which an active site is subdivided into a region of binding and a region of reaction. Analysis of the resulting free energy levels clarifies several points of confusion regarding the nature of enzyme catalysis, including why enzyme/substrate complexes form if, indeed, they only "waste catalytic power". Circumstances are defined in which an evolving enzyme can both lower Km (i.e., enhance substrate binding) and improve the forward catalytic rate while never meddling with the transition structure at the reactive site. It is argued that this process is most advantageously viewed as a substrate destabilization embodying "conserved" interactions at the binding region. Classical transition-state stabilization and an "anti-Pauling" effect are both capable of inducing rate accelerations. In certain circumstances, the latter can predominate as it does with many enzyme-like intramolecular reactions. Behavioral modes discussed herein are applicable to the chemistry of catalytic host/guest and enzyme systems.