Enzyme Intermediates Research Articles

Components of a standardised olive leaf dry extract (Ph. Eur.) promote hypothiocyanite production by lactoperoxidase

We investigated in vitro the ability of a standardised olive leaf dry extract (Ph. Eur.) (OLE) as well as of its single components to circumvent the hydrogen peroxide-induced inhibition of the hypothiocyanite-producing activity of lactoperoxidase (LPO). The rate of hypothiocyanite (−OSCN) formation by LPO was quantified by spectrophotometric detection of the oxidation of 5-thio-2-nitrobenzoic acid (TNB).By using excess hydrogen peroxide, we forced the accumulation of inactive enzymatic intermediates which are unable to promote the two-electronic oxidation of thiocyanate. Both OLE and certain extract components showed a strong LPO-reactivating effect. Thereby an o-hydroxyphenolic moiety emerged to be essential for a good reactivity with the inactive LPO redox states. This basic moiety is found in the main OLE components oleuropein, oleacein, hydroxytyrosol, caffeic acid as well as in different other constituents including the OLE flavone luteolin.As LPO is a key player in the humoral immune response, these results propose a new mode of action regarding the well-known bacteriostatic and anti-inflammatory properties of the leaf extract of Olea europaea L.

Tuning Mechanism-Based Inactivators of Neuraminidases: Mechanistic and Structural Insights

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

Tuning Mechanism‐Based Inactivators of Neuraminidases: Mechanistic and Structural Insights

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

Strong, Low-Barrier Hydrogen Bonds May Be Available to Enzymes

The debate over the possible role of strong, low-barrier hydrogen bonds in stabilizing reaction intermediates at enzyme active sites has taken place in the absence of an awareness of the upper limits to the strengths of low-barrier hydrogen bonds involving amino acid side chains. Hydrogen bonds exhibit their maximal strengths in isolation, i.e., in the gas phase. In this work, we measured the ionic hydrogen bond strengths of three enzymatically relevant model systems in the gas phase using anion photoelectron spectroscopy; we calibrated these against the hydrogen bond strength of HF2(-), measured using the same technique, and we compared our results with other gas-phase experimental data. The model systems studied here, the formate-formic acid, acetate-acetic acid, and imidazolide-imidazole anionic complexes, all exhibit very strong hydrogen bonds, whose strengths compare favorably with that of the hydrogen bifluoride anion, the strongest known hydrogen bond. The hydrogen bond strengths of these gas-phase complexes are stronger than those typically estimated as being required to stabilize enzymatic intermediates. If there were to be enzyme active site environments that can facilitate the retention of a significant fraction of the strengths of these isolated (gas-phase), hydrogen bonded couples, then low-barrier hydrogen bonding interactions might well play important roles in enzymatic catalysis.

Type II Flavin‐Containing Monooxygenases: A New Class of Biocatalysts that Harbors Baeyer–Villiger Monooxygenases with a Relaxed Coenzyme Specificity

AbstractWithin a newly identified set of flavin‐containing monooxygenases (FMOs) from Rhodococcus jostii RHA1, we have identified three monooxygenases (FMO‐E, FMO‐F, and FMO‐G) that are effective in catalyzing Baeyer–Villiger oxidations. These type II FMOs display relaxed coenzyme specificity by accepting both NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and NADH (reduced form of nicotinamide adenine dinucleotide), as a coenzyme, which is a novel and attractive feature among biocatalysts capable of conducting Baeyer–Villiger oxidations. We purified FMO‐E and determined that the Michaelis constants for both coenzymes were in the micromolar range, whereas the activity was highest for NADH. By using the stopped‐flow technique, formation of a peroxyflavin–enzyme intermediate was observed, which indicated that type II FMOs follow a catalytic mechanism similar to that of other class B flavoprotein monooxygenases. A set of cyclobutanones and cyclohexanones were used to probe the regio‐ and enantioselectivity of all three recombinant monooxygenases. The biocatalysts readily accepted small cyclic ketones, which enabled the conversion of previously poorly accepted substrates by other monooxygenases (especially norcamphor), and exhibited excellent and unique regio‐ and enantioselectivities. Sequence analysis revealed that type II FMOs that act as Baeyer–Villiger monooxygenases contain a unique N‐terminal domain. Sequence conservation in this protein domain can be used to identify new NADH‐dependent Baeyer–Villiger monooxygenases, which would facilitate future biocatalyst discovery efforts.

A caged, destabilized, free radical intermediate in the q-cycle.

The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant-induced reduction reaction at their quinol oxidase (Qo ) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one through a series of high-potential electron carriers, the second through low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-cycle. The semiquinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q-cycle semiquinone anion intermediate, termed SQo , in bacterial cytochrome bc1 by rapid freeze-quenching. In this work, we apply pulsed-EPR techniques to determine the location and properties of SQo in the mitochondrial complex. In contrast to semiquinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Qo at a site that is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analyses show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves "stripping" of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Qo proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), conserving redox energy to drive electron transfer to cytochrome bL while minimizing certain Q-cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O2 .

Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon-carbon bond (d>1.6Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate-cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.

Observation of a stable carbene at the active site of a thiamin enzyme

Carbenes are highly reactive chemical compounds that are exploited as ligands in organometallic chemistry and are powerful organic catalysts. They were postulated to occur as transient intermediates in enzymes, yet their existence in a biological system could never be demonstrated directly. We present spectroscopic and structural data of a thiamin enzyme in a noncovalent complex with substrate, which implicate accumulation of a stable carbene as a major resonance contributor to deprotonated thiamin.

Nuclear resonance vibrational spectroscopic and computational study of high-valent diiron complexes relevant to enzyme intermediates

High-valent intermediates of binuclear nonheme iron enzymes are structurally unknown despite their importance for understanding enzyme reactivity. Nuclear resonance vibrational spectroscopy combined with density functional theory calculations has been applied to structurally well-characterized high-valent mono- and di-oxo bridged binuclear Fe model complexes. Low-frequency vibrational modes of these high-valent diiron complexes involving Fe motion have been observed and assigned. These are independent of Fe oxidation state and show a strong dependence on spin state. It is important to note that they are sensitive to the nature of the Fe2 core bridges and provide the basis for interpreting parallel nuclear resonance vibrational spectroscopy data on the high-valent oxo intermediates in the binuclear nonheme iron enzymes.

Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery.

Reactive oxygen, nitrogen, and sulfur species, referred to as ROS, RNS, and RSS, respectively, are produced during normal cell function and in response to various stimuli. An imbalance in the metabolism of these reactive intermediates results in the phenomenon known as oxidative stress. If left unchecked, oxidative molecules can inflict damage on all classes of biological macromolecules and eventually lead to cell death. Indeed, sustained elevated levels of reactive species have been implicated in the etiology (e.g., atherosclerosis, hypertension, diabetes) or the progression (e.g., stroke, cancer, and neurodegenerative disorders) of a number of human diseases.1 Over the past several decades, however, a new paradigm has emerged in which the aforementioned species have also been shown to function as targeted, intracellular second messengers with regulatory roles in an array of physiological processes.2 Against this backdrop, it is not surprising that considerable ongoing efforts are aimed at elucidating the role that these reactive intermediates play in health and disease. Site-specific, covalent modification of proteins represents a prominent molecular mechanism for transforming an oxidant signal into a biological response. Amino acids that are candidates for reversible modification include cysteines whose thiol (i.e., sulfhydryl) side chain is deprotonated at physiological pH, which is an important attribute for enhancing reactivity. While reactive species can modify other amino acids (e.g., histidine, methionine, tryptophan, and tyrosine), this Review will focus exclusively on cysteine, whose identity as cellular target or “sensor” of reactive intermediates is most prevalent and established.3 Oxidation of thiols results in a range of sulfur-containing products, not just disulfide bridges, as typically presented in biochemistry textbooks. An overview of the most relevant forms of oxidized sulfur species found in vivo is presented in Chart 1. Open in a separate window Chart 1 Biologically Relevant Cysteine Chemotypesa aRed, irreversible modifications. Green, unique enzyme intermediates. Note: Additional modifications can form as enzyme intermediates including thiyl radicals, disulfides, and persulfides.

Peroxidase activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular electron transfer

Combined quantum mechanical and molecular mechanical (QM/MM) calculations were used to explore the electron pathway involved in the suicide inactivation of cytochrome P450BM3 from Bacillus megaterium. The suicide inactivation is a common phenomenon observed for heme peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by highly oxidizing enzyme intermediates formed during the catalytic cycle. The selected model was a mutant comprising only the heme domain (CYPBM3 21B3) that had been previously evolved to efficiently catalyze hydroxylation reactions with hydrogen peroxide (H2O2) as electron acceptor. An extensive mapping of residues involved in electron transfer routes was obtained from density functional calculations on activated heme (i.e. Compound I) and selected amino acid residues. Identification of oxidizable residues (electron donors) was performed by selectively activating/deactivating different quantum regions. This method allowed a rational identification of key oxidizable targets in order to replace them for less oxidizable residues by site-directed mutagenesis. The residues W96 and F405 were consistently predicted by the QM/MM electron pathway to hold high spin density; single and double mutants of P450BM3 on these positions (W96A, F405L, W96A/F405L) resulted in a more stable variants in the presence of hydrogen peroxide, displaying a similar reaction rate than P450BM3 21B3. Furthermore, mass spectrometry confirmed these oxidation sites and corroborated the possible routes described by QM/MM electron transfer (ET) pathways.

Synthesis and characterization of new phenyl esters derived from 16-dehydropregnenolone acetate (16-DPA)

A series of new phenyl esters based on a 5,16-pregnadiene-20-one skeleton, namely 3β-benzoyloxy-5,16-pregnadiene-20-ones, which may be good inhibitors of 17α-hydroxylase and 5α-reductase enzyme or useful intermediates for producing steroidal drugs, were synthesized starting from diosgenin. The structures of the steroids were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, infrared (IR), and mass spectra.

Hydrolysis of low concentrations of the acetylthiocholine analogs acetyl(homo)thiocholine and acetyl(nor)thiocholine by acetylcholinesterase may be limited by selective gating at the enzyme peripheral site

Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant kE (often denoted kcat/KM) that approaches 108M−1s−1. AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetyl(homo)thiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetyl(nor)thiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of kE for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The kE for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but kE values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that kE for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site.

Effective methods for preparation and characterization of HNO‐adducts of heme proteins

Driven by the documented effectiveness of cryoradiolysis coupled with resonance Raman spectroscopy for the preparation and structural characterization of unstable peroxo and hydroperoxo intermediates of catalytic and oxidative enzymes, similar strategies have now been applied to prepare and study the still relatively unexplored chemistry of nitrosyl hydride (HNO) adducts of heme proteins and their derivatives. Previously, such HNO derivatives of heme proteins were prepared by chemical addition of reducing agents or by direct addition of an HNO donor. Here, for the first time, we report effective cryoradiolytic preparation of Fe–N(H)O species of myoglobin and their structural characterization by resonance Raman spectroscopy. Our results are in excellent agreement with those previously reported for chemically prepared derivatives and with computational results. Furthermore, the present study provides new data for the deuterated analogue; i.e. bearing a nitrosyl deuteride (DNO) fragment. Copyright © 2012 John Wiley & Sons, Ltd.

Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction

Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD+-dependent steps via covalent thiohemiacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through attack of Cys276 on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu161→Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (⩽2%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by thiohemiacetal adduct formation.

1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were synthesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight binding inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model of inhibition, demonstrating either an encounter complex or a condensation product for the slow and slow-tight binding inhibitors, respectively.

Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates

Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD(+)-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu(161)) by an incompetent analog (Gln(161)) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD(+), we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys(276) was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD(+). The proposed catalytic mechanism of human UGDH involves Lys(220) as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp(280) deprotonates Cys(276) to function as an aldehyde trap and also provides oxyanion stabilization. Glu(161) is the Brønsted base catalytically promoting the thioester hydrolysis.

Revisiting the Peroxidase Oxidation of 2,4,6-Trihalophenols: ESR Detection of Radical Intermediates

The peroxidase oxidation of 2,4,6-trichlorophenol (TCP) has been clearly shown to result in 2,6-dichloro-1,4-benzoquinone (DCQ). DCQ is a 2-electron oxidation product of TCP that has undergone para dechlorination. Many peroxidases show similar oxidation of the substrate, TCP, to yield the quinone, DCQ. Depending on the substrate, peroxidases are thought to carry out both 1- and 2-electron oxidations; the mechanism can be confirmed by the detection of both enzyme and substrate intermediates. This article presents ESR evidence for the transient 2,4,6-trichlorophenoxyl radical intermediate (TCP•), which exists free in solution, i.e., is not enzyme associated. These data are best explained as a 1-electron peroxidase oxidation of TCP to form TCP•, followed by enzyme-independent radical reactions leading to the 2-electron oxidized product. Also presented are data for the peroxidase oxidation of 2,4,6-trifluorophenol and 2,6-dichloro-4-fluorophenol.

Structural and Spectroscopic Characterization of Metastable Thiolate-Ligated Manganese(III)–Alkylperoxo Species

Metastable Mn-peroxo species are proposed to form as key intermediates in biological oxidation reactions involving O(2) and C-H bond activation. The majority of these have yet to be spectroscopically characterized, and their inherent instability, in most cases, precludes structural characterization. Cysteinate-ligated metal-peroxos have been shown to form as reactive intermediates in both heme and nonheme iron enzymes. Herein we report the only examples of isolable Mn(III)-alkylperoxo species, and the first two examples of structurally characterized synthetic thiolate-ligated metal-peroxos. Spectroscopic data, including electronic absorption and IR spectra, and ESI mass spectra for (16)O vs (18)O-labeled metastable Mn(III)-OOR (R = (t)Bu, Cm) are discussed, as well as preliminary reactivity.

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Currently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K(i) values ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C(3) carboxylate of inhibitors and that the localization of this positive charge to a "region of the active site" rather than a specific residue represents an important evolutionary strategy used by β-lactamases.