Substrate Turnover Research Articles

Strict substrate requirements alongside rapid substrate turnover may indicate an early colonization: A case study of Pleurotus calyptratus (Agaricales, Basidiomycota)

Pleurotus calyptratus is a wood-inhabiting oyster mushroom associated with Populus tremula and P. alba . This fungus is rather common in Ukraine, but considered rare in many other European countries. Aiming to find causes for variation in rarity, we conducted habitat mapping and repeated surveys to understand the substrate requirements and fruiting dynamics of P. calyptratus . We showed that fruiting is strictly associated with older trees and early decay stages, and not with physical properties of a tree trunk like volume or position. This timespan of substrate suitability lasts for 1–3 y, determining rapid substrate turnover. We also detected DNA of P. calyptratus in living tissues of visually healthy trees, meaning its latent presence in the hosts, which places the fungus in a position to be a pioneer in a fungal succession. Despite the high spatial and temporal variation in fruiting, five annual surveys demonstrated no significant trends in P. calyptratus population's dynamics, and supported the assumption of random variation in fruiting, driven mostly by dynamics of forest stands. Therefore, we hypothesize that P. calyptratus ' relative abundance in Ukraine is explained by continuous substrate supply from large homogenous aspen stands that are rare in the rest of Europe due to intensive management. • Pleurotus calyptratus shows yet unexplained differences in rarity across Europe. • Repeated fruit body surveys were applied to explore species population dynamics. • Latent development of P. calyptratus in young living trees has been proven. • P. calyptratus combines both strict requirements and rapid turnover of substrates. • Such fruiting pattern can indicate an early colonization strategy for agarics.

Reprograming of proteasomal degradation by branched chain amino acid metabolism.

Branched-chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi-omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin-proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP-based proteasome substrate, which is suppressed by loss-of-function of the first BCAA catabolic enzyme, the branched-chain aminotransferase BCAT-1. The exogenous supply of BCAA-derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.

YfeX - A New Platform for Carbene Transferase Development with High Intrinsic Reactivity.

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS‐CoV‐2

Monoclonal antibodies targeting the SARS‐CoV‐2 spike (S) neutralize infection and are efficacious for the treatment of COVID‐19. However, SARS‐CoV‐2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS‐CoV‐2 variants and related betacoronaviruses. The high‐affinity and catalytically active decoy sACE22.v2.4‐IgG1 was previously shown to be effective against SARS‐CoV‐2 variants when administered intravenously. Here, inhalation of aerosolized sACE22.v2.4‐IgG1 increased survival and ameliorated lung injury in K18‐hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22.v2.4‐IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18‐hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22.v2.4‐IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS‐CoV‐2 variants.

A structural basis for the functional differences between the cytosolic and plastid phosphoglucose isomerase isozymes.

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), thereby regulating sucrose synthesis in plant cells. In general, plants contain a pair of PGI isozymes located in two distinct compartments of the cell (cytosol and plastid) with differences in both the primary structure and the higher-order structure. Previously, we showed that the activity of cytosolic PGI (PGIc) is more robust (activity, thermal stability, substrate turnover rate, etc.) than that of the plastid counterpart (PGIp) in multiple organisms, including wheat, rice, and Arabidopsis. The crystal structures of apoTaPGIc (an isotype cytosol PGIc in Triticum aestivum), TaPGIc-G6P complex, and apoTaPGIp (an isotype plastid PGIp in Triticum aestivum) were first solved in higher plants, especially in crops. In this study, we detailed the structural characteristics related to the biochemical properties and functions of TaPGIs in different plant organelles. We found that the C-terminal domains (CTDs) of TaPGIc and TaPGIp are very different, which affects the stability of the dimerized enzyme, and that Lys213TaPGIc/Lys193TaPGIp and its surrounding residues at the binding pocket gateway may participate in the entrance and exit of substrates. Our findings provide a good example illuminating the evolution of proteins from primary to higher structures as a result of physical barriers and adaptation to the biochemical environment.

The coupling of mitoproteolysis and oxidative phosphorylation enables tracking of an active mitochondrial state through MitoTimer fluorescence

The regulation of mitochondria function and health is a central node in tissue maintenance, ageing as well as the pathogenesis of various diseases. However, the maintenance of an active mitochondrial functional state and its quality control mechanisms remain incompletely understood. By studying mice with a mitochondria-targeted reporter that shifts its fluorescence from “green” to “red” with time (MitoTimer), we found MitoTimer fluorescence spectrum was heavily dependent on the oxidative metabolic state in the skeletal muscle fibers. The mitoproteolytic activity was enhanced in an energy dependent manner, and accelerated the turnover of MitoTimer protein and respiratory chain substrate, responsible for a green predominant MitoTimer fluorescence spectrum under the oxidative conditions. PGC1α, as well as anti-ageing regents promoted enhanced mitoproteolysis. In addition, cells with the green predominant mitochondria exhibited lower levels of MitoSox and protein carbonylation, indicating a favorable redox state. Thus, we identified MitoTimer as a probe for mitoproteolytic activity in vivo and found a heightened control of mitoproteolysis in the oxidative metabolic state, providing a framework for understanding the maintenance of active oxidative metabolism while limiting oxidative damages.

Two-Phase Conceptual Framework of Phosphatase Activity and Phosphorus Bioavailability.

The activity of extracellular phosphatases is a dynamic process controlled by both plant roots and microorganisms, which is responsible for the mineralization of soil phosphorus (P). Plants regulate the availability of soil P through the release of root mucilage and the exudation of low-molecular weight organic acids (LMWOAs). Mucilage increases soil hydraulic conductivity as well as pore connectivity, both of which are associated with increased phosphatase activity. The LMWOAs, in turn, stimulate the mineralization of soil P through their synergistic effects of acidification, chelation, and exchange reactions. This article reviews the catalytic properties of extracellular phosphatases and their interactions with the rhizosphere interfaces. We observed a biphasic effect of root metabolic products on extracellular phosphatases, which notably altered their catalytic mechanism. In accordance with the proposed conceptual framework, soil P is acquired by both plants and microorganisms in a coupled manner that is characterized by the exudation of their metabolic products. Due to inactive or reduced root exudation, plants recycle P through adsorption on the soil matrix, thereby reducing the rhizosphere phosphatase activity. The two-phase conceptual framework might assist in understanding P-acquisition (substrate turnover) and P-restoration (phosphatase adsorption by soil) in various terrestrial ecosystems.

Sequence dependencies and biophysical features both govern cleavage of diverse cut-sites by HIV protease.

The infectivity of HIV-1 requires its protease (PR) cleave multiple cut-sites with low sequence similarity. The diversity of cleavage sites has made it challenging to investigate the underlying sequence properties that determine binding and turnover of substrates by PR. We engineered a mutational scanning approach utilizing yeast display, flow cytometry, and deep sequencing to systematically measure the impacts of all individual amino acid changes at 12 positions in three different cut-sites (MA/CA, NC/p1, and p1/p6). The resulting fitness landscapes revealed common physical features that underlie cutting of all three cut-sites at the amino acid positions closest to the scissile bond. In contrast, positions more than two amino acids away from the scissile bond exhibited a strong dependence on the sequence background of the rest of the cut-site. We observed multiple amino acid changes in cut-sites that led to faster cleavage rates, including a preference for negative charge five and six amino acids away from the scissile bond at locations where the surface of protease is positively charged. Analysis of individual cut sites using full-length matrix-capsid proteins indicate that long-distance sequence context can contribute to cutting efficiency such that analyses of peptides or shorter engineered constructs including those in this work should be considered carefully. This work provides a framework for understanding how diverse substrates interact with HIV-1 PR and can be extended to investigate other viral PRs with similar properties.

Optimizing Efficiency and Motility of a Polyvalent Molecular Motor.

Molecular motors play a vital role in the transport of material within the cell. A family of motors of growing interest are burnt bridge ratchets (BBRs). BBRs rectify spatial fluctuations into directed motion by creating and destroying motor-substrate bonds. It has been shown that the motility of a BBR can be optimized as a function of the system parameters. However, the amount of energy input required to generate such motion and the resulting efficiency has been less well characterized. Here, using a deterministic model, we calculate the efficiency of a particular type of BBR, namely a polyvalent hub interacting with a surface of substrate. We find that there is an optimal burn rate and substrate concentration that leads to optimal efficiency. Additionally, the substrate turnover rate has important implications on motor efficiency. We also consider the effects of force-dependent unbinding on the efficiency and find that under certain conditions the motor works more efficiently when bond breaking is included. Our results provide guidance for how to optimize the efficiency of BBRs.

Functional Characterisation of Two Novel Deacetylases from Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) is an exclusively human pathogen that causes a wide range of diseases. We have identified two novel proteins, Spy1094 and Spy1370, which show sequence similarity with peptidoglycan deacetylases (PGDAs) from other streptococcal species like S. pneumoniae and S. iniae, that represent important virulence factors. Recombinant Spy1094 and Spy1370 were active at a wide pH range (pH 4.0–9.0) and showed metal ion-dependence, with the highest activities observed in the presence of Mn2+, Mg2+and Zn2+. The enzymes showed typical Michaelis–Menten saturation kinetics with the pseudo-substrate GlcNAc3. Binding affinities for rSpy1094 and rSpy1370 were high (Km = 2.2 ± 0.9 μM and 3.1 ± 1.1 μM, respectively), but substrate turnover was low (Kcat = 0.0075/s and 0.0089/s, respectively) suggesting that peptidoglycan might not be the preferred target for deacetylation. Both enzymes were expressed during bacterial growth.

Mutualistic interaction between arbuscular mycorrhiza fungi and soybean roots enhances drought resistant through regulating glucose exudation and rhizosphere expansion

Glucose is one of the low molecular weight components of root exudates to mediate the cross-talk between plants and microbes, but less is known about their contribution to drought resistance of plants and root-associated microbiome. To fill this knowledge gap, we optimized the visualization of glucose exudation and coupled it with another in situ tool – soil zymography ‒ as well as destructive analysis of enzyme kinetics (β-glucosidase; acid phosphomonoesterase) and microbial biomass. This helped identify how microbial functionality ‒ affected by drought and P limitation ‒ will show more resistance in the hotspots of soybean rhizosphere (grown in the rhizoboxes for 10 weeks) associated with arbuscular mycorrhizal fungi (AMF) symbiosis than those without AMF. Drought reduced glucose exudation, mainly allocated to root tips, and narrowed the rhizosphere enzymatic hotspot by three times. However, AMF inoculation enhanced glucose exudation compared to non-mycorrhizal plants and enlarged enzymatic hotspot area by 53% under drought condition. Despite the 50% reduction in β-glucosidase and acid phosphomonoesterase activities owing to water deficit, AMF symbiont triggered up to 36% enzyme activities in correlation with the non-mycorrhizal ones. Therefore, the drought resistance of these two enzymes was enhanced by up to 63% in mycorrhizal plants. The biomass of microbial phosphorus increased by 45% under drought AMF-conditioned plants. We conclude that the cooperation between soybean and AMF induced the formation of favorable microsites around the root, specifically in overlapping localities between rhizosphere and mycorrhizosphere, characterized by enhanced glucose release, increasing rhizosphere expansion, high enzyme activities and shortened substrate turnover time. This, in turn, contributed to the stronger resistance of microbial functions (e.g., enzyme expression) to drought stress in the rhizosphere hotspots. Thus, in response to AMF inoculation and consequent high glucose availability, rhizosphere microorganisms increased P mining rate in those hotspots remaining active despite water scarcity.

Changes in the Left Ventricular Eicosanoid Profile in Human Dilated Cardiomyopathy.

ObjectiveMetabolites derived from N−3 and N−6 polyunsaturated fatty acids (PUFAs) have both beneficial and detrimental effects on the heart. However, contribution of these lipid mediators to dilated cardiomyopathy (DCM)-associated mitochondrial dysfunction remains unknown. This study aimed to characterize DCM-specific alterations in the PUFA metabolome in conjunction with cardiac mitochondrial quality in human explanted heart tissues.MethodsLeft ventricular tissues obtained from non-failing control (NFC) or DCM explanted hearts, were assessed for N−3 and N−6 PUFA metabolite levels using LC-MS/MS. mRNA and protein expression of CYP2J2, CYP2C8 and epoxide hydrolase enzymes involved in N−3 and N−6 PUFA metabolism were quantified. Cardiac mitochondrial quality was assessed by transmission electron microscopy, measurement of respiratory chain complex activities and oxygen consumption (respiratory control ratio, RCR) during ADP-stimulated ATP production.ResultsFormation of cardioprotective CYP-derived lipid mediators, epoxy fatty acids (EpFAs), and their corresponding diols were enhanced in DCM hearts. These findings were corroborated by increased expression of CYP2J2 and CYP2C8 enzymes, as well as microsomal and soluble epoxide hydrolase enzymes, suggesting enhanced metabolic flux and EpFA substrate turnover. DCM hearts demonstrated marked damage to mitochondrial ultrastructure and attenuated mitochondrial function. Incubation of fresh DCM cardiac fibers with the protective EpFA, 19,20-EDP, significantly improved mitochondrial function.ConclusionsThe current study demonstrates that increased expressions of CYP-epoxygenase enzymes and epoxide hydrolases in the DCM heart correspond with enhanced PUFA-derived EpFA turnover. This is accompanied by severe mitochondrial functional impairment which can be rescued by the administration of exogenous EpFAs.

Fertilization promotes microbial growth and minimum tillage increases nutrient-acquiring enzyme activities in a semiarid agro-ecosystem

Microorganisms respiratory and enzymatic activities provide sensitive indicators of changes in soil properties, such as those caused by interactive effects of tillage and fertilization regimes or other agricultural practices. However, the rapid, adaptive microbial growth, respiratory and enzymatic responses to changes in soil environments induced by specific agricultural practices are not well understood. Thus, to explore these adaptations we compared effects of contrasting environments on functional microbial traits (growth and enzyme kinetic parameters) in a Mediterranean agro-ecosystem. These environments differed in long-term disturbance (no, minimum, or conventional tillage), nitrogen-richness (fertilization with 90 kg N ha−1 versus no fertilization), and resource scarcity (increasing with soil depth in 0–30, 30–60 and 60–90 cm layers). Reducing soil disturbance from conventional to minimum tillage promoted microbial growth through shorter Tlag and larger active biomass fraction and induced increases in N- and P-acquiring enzyme activities by increasing nutrients limitation. Fertilization stimulated increases in fast-growing microorganisms with low substrate-affinity enzyme systems, microbial biomass, enzymatic activities, and turnover rates of soil organics. In contrast, increasing scarcity of resources with soil depth strongly reduced microbial biomass and activity. A lack of correlation between soil and enzymatic stoichiometric ratios raises concern regarding the applicability of eco-enzymatic stoichiometric indexes in Mediterranean agro-ecosystems. We conclude that decomposition and turnover of organic substrates under contrasting agricultural practices are mediated by microbial communities with distinct functional traits (active fraction, growth parameters) and enzyme properties (Vmax, Km), which need to be considered in smart land use regimes.

On the kinetic mechanism of dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18) catalyzes the hydrolysis of asymmetric Nω,Nω-dimethyl-l-arginine (ADMA), an endogenous inhibitor of human nitric oxide synthases. The active-site cysteine residue has been proposed to serve as the catalytic nucleophile, forming an S-alkylthiourea reaction intermediate, and serving as a target for covalent inhibitors. Inhibition can lead to ADMA accumulation and downstream inhibition of nitric oxide production. Prior studies have provided experimental evidence for formation of this covalent adduct but have not characterized it kinetically. Here, rapid quench-flow is used with ADMA and the DDAH from Pseudomonas aeruginosa to determine the rate constants for formation (k2 = 17 ± 2 s−1) and decay (k3 = 1.5 ± 0.1 s−1) of the covalent S-alkylthiourea adduct. A minimal kinetic mechanism for DDAH is proposed that supports the kinetic competence of this species as a covalent reaction intermediate and assigns the rate-limiting step in substrate turnover as hydrolysis of this intermediate. This work helps elucidate the different reactivities of S-alkylthiourea intermediates found among the mechanistically diverse pentein superfamily of guanidine-modifying enzymes and provides information useful for inhibitor development.

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.

Occurrence of a novel cleavage site for cathepsin G adjacent to the polybasic sequence within the proteolytically sensitive activation loop of the SARS-CoV-2 Omicron variant: The amino acid substitution N679K and P681H of the spike protein

The serine proteases neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and neutrophil serine protease 4 (NSP4) are secreted by activated neutrophils as a part of the innate immune response against invading pathogens. However, these serine proteases might be adopted by viruses to mediate viral surface protein priming resulting in host cell entrance and productive infection. Indeed, NE and PR3 hydrolyze the scissile peptide bond within the proteolytically sensitive polybasic sequence of the activation loop of SARS-CoV-2 located at the S1/S2 interface of the Spike (S) protein; an amino acid motif which differs from SARS-CoV-1. The occurrence of novel SARS-CoV-2 variants and substitution of distinct amino acids at the polybasic sequence prompts serious concerns regarding increased transmissibility. We propose that a novel cleavage site by CatG of the Omicron variant and the increased substrate turnover of the Delta variant by furin within the polybasic sequence should be considered for increased transmission of SARS-CoV-2 variants.

Unfolding by Cdc48/p97: different strokes for different folks.

The protein unfoldase Cdc48/p97 targets a wide variety of cellular substrates, but the molecular basis of substrate turnover remains incompletely understood. Two recent reports, by Ji et al. and van den Boom et al., provide detailed insights into the unfolding process and reveal pronounced flexibility of substrate handling by Cdc48/p97.

Enzymatic Epoxidation of Long-Chain Terminal Alkenes by Fungal Peroxygenases.

Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, flavoring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosubstrate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosubstrate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, notable differences were observed between them, with rCciUPO being responsible for the highest substrate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies.

An improved Amplex Red‐based fluorometric assay of phenol oxidases and peroxidases activity: A case study on Haplic Chernozem

AbstractThe initial steps of complex organic matter degradation in soil comprise several oxidative processes catalysed by phenol oxidases and peroxidases. Commonly, their activity is determined by colorimetric approaches based on a range of substrates, making comparison between studies difficult. To improve this shortcoming, we conducted a calibration procedure for assessing phenol oxidase and peroxidase activities in soils by a sensitive fluorometric assay. This assay is based on the colourless and non‐fluorescent Amplex Red® reagent as a substrate, which forms a fluorescent product, resorufin, upon oxidation. Commercially available resorufin was used as a standard for calibration. We applied Michaelis–Menten kinetics to determine functional traits of phenol oxidases and peroxidases in a case study on Haplic Chernozem from different land‐use and climate treatments of a field experiment. The kinetic parameters, limit of detection and limit of quantification demonstrated a high sensitivity of the fluorometric assay to differences caused by these treatments. For the total oxidative enzymes, the catalytic efficiency (Kcat, calculated as Vmax/Km) was 30% higher in the cropland compared with the pasture plot. However, compared with soil samples collected from ambient climatic conditions, Kcat of total oxidoreductases was 60% lower in cropland exposed to elevated air temperature and drought. The phenol oxidase activity was substantially higher than peroxidase activity in cropland land use. However, higher Kcat of peroxidase than phenol oxidase resulted in 60% faster substrate turnover under both climatic conditions. In our case study, the Amplex Red‐based assay enabled rapid and high‐throughput analyses of phenol oxidase and peroxidase activity in soil. The sensitivity of this assay for soil enzymology applications needs further testing for a larger range of soils and environmental conditions.Highlights A novel fluorometric assay of phenol oxidase and peroxidase activity in soil is proposed Resorufin fits very well for calibration simplifying the comparisons between studies Phenol oxidase and peroxidase displayed apparent Michaelis–Menten kinetics Amplex Red was sensitive for land‐use and climate treatments on Haplic Chernozem

The ATP/Mg2+ Balance Affects the Degradation of Short Fluorogenic Substrates by the 20S Proteasome

Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg2+, and DTT. ATP and Mg2+ are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg2+ on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg2+. Moreover, the substrate degradation efficacy was proportional to the Mg2+/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg2+ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.