The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.     

Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases

Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by carrying out a detailed investigation of the enzymatic reaction mechanism. This investigation included the X-ray determination of 15 crystal structures of two homologous enzymes in free and complexed states (with inhibitors) and extensive ab initio calculations of the interactions between the active sites and the reaction intermediates. The catalytic residues, through short strong hydrogen bonds, play the role of charge buffer to stabilize the negative charge built up on the intermediates in the course of the reaction. The hydrogen bond distances in the intermediate analogues are found to be about 0.2 A shorter in the product analogues both experimentally and theoretically.     

NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.     

Mechanistic Insights into Ferredoxin-NADP(H) Reductase Catalysis Involving the Conserved Glutamate in the Active Site

Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results.     

The dynamic role of distal side residues in heme hydroperoxidase catalysis. Interplay between X-ray crystallography and ab initio MD simulations

The enzymatic cycle of hydroperoxidases involves the resting Fe(III) state of the enzyme and the high-valent iron intermediates Compound I and Compound II. These states might be characterized by X-ray crystallography and the transition pathways between each state can be investigated using atomistic simulations. Here we review our recent work in the modeling of two key steps of the enzymatic reaction of hydroperoxidases: the formation of Cpd I in peroxidase and the reduction of Cpd I in catalase. It will be shown that small conformational motions of distal side residues (His in peroxidases and His/Asn in catalases), not,or only partially, revealed by the available X-ray structures, play an important role in the catalytic processes examined.     

Crystal structures of the reaction intermediate and its homologue of an extradiol-cleaving catecholic dioxygenase

BphC derived from Pseudomonas sp. strain KKS102 is an extradiol-cleaving catecholic dioxygenase. This enzyme contains a non-heme iron atom and plays an important role in degrading biphenyl/polychlorinated biphenyls (PCBs) in the microbe. To elucidate detailed structures of BphC reaction intermediates, crystal structures of the substrate-free form, the BphC-substrate complex, and the BphC-substrate-NO (nitric oxide) complex were determined. These crystal structures revealed (1) the binding site of the O(2) molecule in the coordination sphere and (2) conformational changes of His194 during the catalytic reaction. On the basis of these findings, we propose a catalytic mechanism for the extradiol-cleaving catecholic dioxygenase in which His194 seems to play three distinct roles. At the early stage of the catalytic reaction, His194 appears to act as a catalytic base, which likely deprotonates the hydroxyl group of the substrate. At the next stage, the protonated His194 seems to stabilize a negative charge on the O2 molecule located in the hydrophobic O2-binding cavity. Finally, protonated His194 seems to function as a proton donor, whose existence has been proposed.     

Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.     

Rational Design of Hybrid Nanostructures for Advanced Photocatalysis

Nanocatalysis has been a growing field over the past few decades with significant developments in understanding the surface properties of nanocatalysts. With recent advances in synthetic methods, size, shape and composition of the nanoparticles can be controlled in a well defined manner which facilitates achieving selective reaction products in multipath reactions. Nanoparticles with specific exposed crystal facets can have different reactivity than other facets for reaction intermediates, which favours selective pathways during the course of reaction. Heterogeneous catalysts have been studied extensively; nano‐sized metal particles are absorbed on mesoporus supports, facilitating access to the large surface area of the nanoparticles and hence exposure of more catalytic sites. Photocatalysis is attractive area of catalysis, in which photoinduced charge carriers are used for a variety of catalytic applications. More interestingly, clean and renewable liquid fuels energy sources such as hydrogen and methyl alcohol can be generated using photocatalysts through water splitting and CO₂ reduction, respectively. Herein, we highlight the progress of nanocatalysis through metal, bimetallic nanoparticle, metal‐semiconductor hybrid nanostructures and oxide nanoparticles for various reactions.     

One- and two-electron oxidation of thiols: mechanisms,kinetics and biological fates

The oxidation of biothiols participates not only in the defense against oxidative damage but also in enzymatic catalytic mechanisms and signal transduction processes. Thiols are versatile reductants that react with oxidizing species by one- and two-electron mechanisms, leading to thiyl radicals and sulfenic acids, respectively. These intermediates, depending on the conditions, participate in further reactions that converge on different stable products. Through this review, we will describe the biologically relevant species that are able to perform these oxidations and we will analyze the mechanisms and kinetics of the one- and two-electron reactions. The processes undergone by typical low-molecular-weight thiols as well as the particularities of specific thiol proteins will be described, including the molecular determinants proposed to account for the extraordinary reactivities of peroxidatic thiols. Finally, the main fates of the thiyl radical and sulfenic acid intermediates will be summarized.     

Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes — a comparative study of group-I intron structures

In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.     

The tetrahymena ribozyme cleaves a 5'-methylene phosphonate monoester approximately 10(2)-fold faster than a normal phosphate diester: implications for enzyme catalysis of phosphoryl transfer reactions

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by <5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.     

Misunderstanding the preorganization concept can lead to confusions about the origin of enzyme catalysis

Understanding the origin of the catalytic power of enzymes has both conceptual and practical importance. One of the most important finding from computational studies of enzyme catalysis is that a major part of the catalytic power is due to the preorganization of the enzyme active site. Unfortunately, misunderstanding of the nontrivial preorganization idea lead some to assume that it does not consider the effect of the protein residues. This major confusion reflects a misunderstanding of the statement that the interaction energy of the enzyme group and the transition state (TS) is similar to the corresponding interaction between the water molecules (in the reference system) and the TS, and that the catalysis is due to the reorganization free energy of the water molecules. Obviously, this finding does not mean that we do not consider the enzyme groups. Another problem is the idea that catalysis is due to substrate preorganization. This more traditional idea is based in some cases on inconsistent interpretation of the action of model compounds, which unfortunately, do not reflect the actual situation in the enzyme active site. The present article addresses the above problems, clarifying first the enzyme polar preorganization idea and the current misunderstandings. Next we take a specific model compound that was used to promote the substrate preorganization proposal and establish its irrelevance to enzyme catalysis. Overall, we show that the origin of the catalytic power of enzymes cannot be assessed uniquely without computer simulations, since at present this is the only way of relating structure and energetics.     

Investigation of the role of the histidine-aspartate pair in the human exonuclease III-like abasic endonuclease,Ape1

Hydrogen bonded histidine-aspartate (His-Asp) pairs are critical constituents in several key enzymatic reactions. To date, the role that these pairs play in catalysis is best understood in serine and trypsin-like proteases, where structural and biochemical NMR studies have revealed important pK(a) values and hydrogen bonding patterns within the catalytic pocket. However, the role of the His-Asp pair in metal-assisted catalysis is less clear. Here, we apply liquid-state NMR to investigate the role of a critical histidine residue of apurinic endonuclease 1 (Ape1), a human DNA repair enzyme that cleaves adjacent to abasic sites in DNA using one or more divalent cations and an active-site His-Asp pair. The results of these studies suggest that the Ape1 His-Asp pair does not function as either a general base catalyst or a metal ligand. Rather, the pair likely stabilizes the pentavalent transition state necessary for phospho-transfer.     

Hydrogenases: active site puzzles and progress

Recent research on the hydrogenase reactions has sought to probe beyond the information that is provided by X-ray diffraction structures. The major challenge of locating 'transient' hydrogen atoms in species that are potential catalytic intermediates is being addressed, using advanced electron paramagnetic resonance (EPR) techniques and theoretical methods. This article discusses recent progress towards a consensus on the structures of different states of the active site of hydrogenases, the mechanisms of activation and hydrogen cycling.     

Short, strong hydrogen bonds at the active site of human acetylcholinesterase: proton NMR studies

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.     

Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.     

High-valent nonheme iron-oxo species in biomimetic oxidations

High valent iron-oxo species are often invoked as the key oxidizing agents in the catalytic cycles of oxygen activating nonheme iron enzymes, and three of these intermediates have in fact been characterized. To gain further insight into such species, a number of biomimetic complexes have been designed and investigated as functional models for these enzymes. Progress since 2000 is summarized in this review. Many of the model complexes discussed in this review carry out oxidative transformations of relevance to the enzymatic reactions; however, the participation of a high-valent iron-oxo species (Fe(IV)O or Fe(V)O) can only be inferred. Arguments in support of a metal-based oxidant (rather than an oxygen radical species) usually hinge on the high conversion for the transformation and the nature of the reaction products, as well as the incorporation of label into these products from H(2)(18)O or related species. Within this time period, the first bona fide nonheme Fe(IV)O complexes have been generated and identified spectroscopically, three of which are crystallographically characterized. Taken together, these studies emphasize the important role the supporting polydentate ligand plays in eliciting the desired high-valent iron-oxo chemistry.     

Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory.