Restricted Substrate Research Articles

Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs)

Glutamate elicits Ca2+ signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca2+ activates respiration by stimulation of the NADH malate–aspartate shuttle through Ca2+-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca2+ binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca2+ entry into the matrix through the mitochondrial Ca2+ uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca2+ deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD+, a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca2+-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca2+ entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca2+, resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/− mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Halanaeroarchaeum sulfurireducens gen. nov., sp. nov., the first obligately anaerobic sulfur-respiring haloarchaeon, isolated from a hypersaline lake

Anaerobic enrichments with acetate as electron donor and carbon source, and elemental sulfur as electron acceptor at 4 M NaCl using anaerobic sediments and brines from several hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in isolation in pure culture of four strains of obligately anaerobic haloarchae growing exclusively by sulfur respiration. Such metabolism has not yet been demonstrated in any known species of Halobacteria, and in the whole archaeal kingdom, acetate oxidation with sulfur as acceptor was not previously demonstrated. The four isolates had nearly identical 16S rRNA gene sequences and formed a novel genus-level branch within the family Halobacteriaceae. The strains had a restricted substrate range limited to acetate and pyruvate as electron donors and elemental sulfur as electron acceptor. In contrast to aerobic haloarchaea, the biomass of anaerobic isolates completely lacked the typical red pigments. Growth with acetate+sulfur was observed between 3-5 M NaCl and at a pH range from 6.7 to 8.0. The membrane core lipids were dominated by archaeols. On the basis of distinct physiological and phylogenetic data, the sulfur-respiring isolates represent a novel species of a new genus in the family Halobacteriaceae, for which the name Halanaeroarchaeaum sulfurireducens gen. nov., sp. nov. is proposed. The type strain of the type species is HSR2T (=JCM 30661T=UNIQEM U935T).

Discovery of a Promiscuous Non‐Heme Iron Halogenase in Ambiguine Alkaloid Biogenesis: Implication for an Evolvable Enzyme Family for Late‐Stage Halogenation of Aliphatic Carbons in Small Molecules

AbstractThe elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non‐heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late‐stage aliphatic C−H group functionalization. Cross‐comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C‐terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild‐type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole‐type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late‐stage modification of unactivated aliphatic carbon centers in small molecules with halogens.

Discovery of a Promiscuous Non-Heme Iron Halogenase in Ambiguine Alkaloid Biogenesis: Implication for an Evolvable Enzyme Family for Late-Stage Halogenation of Aliphatic Carbons in Small Molecules.

The elucidation of enigmatic enzymatic chlorination timing in ambiguine indole alkaloid biogenesis led to the discovery and characterization of AmbO5 protein as a promiscuous non-heme iron aliphatic halogenase. AmbO5 was shown capable of selectively modifying seven structurally distinct ambiguine, fischerindole and hapalindole alkaloids with chlorine via late-stage aliphatic C-H group functionalization. Cross-comparison of AmbO5 with a previously characterized aliphatic halogenase homolog WelO5 that has a restricted substrate scope led to the identification of a C-terminal sequence motif important for substrate tolerance and specificity. Mutagenesis of 18 residues of WelO5 within the identified sequence motif led to a functional mutant with an expanded substrate scope identical to AmbO5, but an altered substrate specificity from the wild-type enzymes. These observations collectively provide evidence on the evolvable nature of AmbO5/WelO5 enzyme duo in the context of hapalindole-type alkaloid biogenesis and implicate their promise for the future development of designer biocatalysis for the selective late-stage modification of unactivated aliphatic carbon centers in small molecules with halogens.

Expanding Beyond the Micropore: Active-Site Engineering in Hierarchical Architectures for Beckmann Rearrangement

The ability to engineer discrete solid-acid centers within hierarchically porous architectures that contain micropores with interconnected mesopores offers the potential to overcome the spatial restraints and diffusional limitations imposed by conventional microporous zeotype catalysts, which often lead to decreased catalyst lifetimes and restricted substrate scope. By employing a one-step soft-templating approach, coupled with detailed physicochemical and spectroscopic characterization, isolated, solid-acid sites can be suitably tailored and discretely modulated within the micropores and mesopores. The design strategy facilitates a synergistic enhancement in catalytic activity, selectivity, substrate versatility, and longevity, compared to analogous microporous zeotypes that have been extensively employed in the chemical industry, as solid-acid catalysts, for the production of e-caprolactam (precursor for Nylon-6).

Palladium-catalyzed aryl C-H olefination with unactivated, aliphatic alkenes.

Palladium-catalyzed coupling between aryl halides and alkenes (Mizoroki-Heck reaction) is one of the most popular reactions for synthesizing complex organic molecules. The limited availability, problematic synthesis, and higher cost of aryl halide precursors (or their equivalents) have encouraged exploration of direct olefination of aryl carbon-hydrogen (C-H) bonds (Fujiwara-Moritani reaction). Despite significant progress, the restricted substrate scope, in particular noncompliance of unactivated aliphatic olefins, has discouraged the use of this greener alternative. Overcoming this serious limitation, we report here a palladium-catalyzed chelation-assisted ortho C-H bond olefination of phenylacetic acid derivatives with unactivated, aliphatic alkenes in good to excellent yields with high regio- and stereoselectivities. The versatility of this operationally simple method has been demonstrated through drug diversification and sequential C-H olefination for synthesizing divinylbenzene derivatives.

Gypsophile chemistry unveiled: Fourier transform infrared (FTIR) spectroscopy provides new insight into plant adaptations to gypsum soils.

Gypsum soils are among the most restrictive and widespread substrates for plant life. Plants living on gypsum are classified as gypsophiles (exclusive to gypsum) and gypsovags (non-exclusive to gypsum). The former have been separated into wide and narrow gypsophiles, each with a putative different ecological strategy. Mechanisms displayed by gypsum plants to compete and survive on gypsum are still not fully understood. The aim of this study was to compare the main chemical groups in the leaves of plants with different specificity to gypsum soils and to explore the ability of Fourier transform infrared (FTIR) spectra analyzed with neural network (NN) modelling to discriminate groups of gypsum plants. Leaf samples of 14 species with different specificity to gypsum soils were analysed with FTIR spectroscopy coupled to neural network (NN) modelling. Spectral data were further related to the N, C, S, P, K, Na, Ca, Mg and ash concentrations of samples. The FTIR spectra of the three groups analyzed showed distinct features that enabled their discrimination through NN models. Wide gypsophiles stood out for the strong presence of inorganic compounds in their leaves, particularly gypsum and, in some species, also calcium oxalate crystals. The spectra of gypsovags had less inorganic chemical species, while those of narrow gypsum endemisms had low inorganics but shared with wide gypsophiles the presence of oxalate. Gypsum and calcium oxalate crystals seem to be widespread amongst gypsum specialist plants, possibly as a way to tolerate excess Ca and sulphate. However, other mechanisms such as the accumulation of sulphates in organic molecules are also compatible with plant specialization to gypsum. While gypsovags seem to be stress tolerant plants that tightly regulate the uptake of S and Ca, the ability of narrow gypsum endemisms to accumulate excess Ca as oxalate may indicate their incipient specialization to gypsum.

Photochemical method for decoration of silver nanoparticles on filter paper substrate for SERS application

A facile photoreduction strategy to attach silver nanoparticles on filter paper (AgNPs@FP) to form surface‐enhanced Raman scattering (SERS) substrate array was proposed and examined. As prepared, SERS substrate array was applied to detect low concentration of adenine and other nucleobases in aqueous solutions. In addition, the formed SERS substrate array allows mass analysis of aqueous samples with a requirement of only 10 μL in sample volume. To optimize and understand the parameters in preparations, factors such as the concentrations of citrate and silver nitrate, photoreduction time, and concentration of sodium hydroxide were varied and examined. Para‐nitrothiophenol was used as a Raman probe molecule and scanning electron microscope (SEM) images of the substrates were used to explore the influences of experimental factors in the preparation of SERS array. Results indicated that silver ions could be effectively reduced and deposited in/on filter paper with the presence of citrate. The formed AgNPs@FP exhibits a three‐dimensional structure as the particles formed on and beneath the surface of the cellulosic fibers could be observed. Deposition of analyte on restricted substrate array rendered reproducible results and the spot‐to‐spot SERS intensity varied within 8%. At optimal conditions, the substrate enhancement factor approached 107. We demonstrated that the filter paper‐based SERS substrates can be used as a suitable tool for biological analysis, and a limit of detection better than 160 nM was obtained in detection of adenine molecules in aqueous solutions with a linear range up to 20 μM. Copyright © 2014 John Wiley & Sons, Ltd.

Synthesis of a novel legumain-cleavable colchicine prodrug with cell-specific toxicity

Conventional chemotherapy has undesirable toxic side-effects to healthy tissues due to low cell selectivity of cytotoxic drugs. One approach to increase the specificity of a cytotoxic drug is to make a less toxic prodrug which becomes activated at the tumour site. The cysteine protease legumain have remarkable restricted substrate specificity and is the only known mammalian asparaginyl (Asn) endopeptidase. Over-expression of legumain is reported in cancers and unstable atherosclerotic plaques, and utilizing legumain is a promising approach to activate prodrugs.In this study we have synthesized the legumain-cleavable peptide sequence N-Boc-Ala-Ala-Asn-Val-OH. The peptide was subsequently conjugated to deacetyl colchicine during three steps to produce Suc-Ala-Ala-Asn-Val-colchicine (prodrug) with >90% chemical purity. Several cell lines with different expressions and activities of legumain were used to evaluate the general toxicity, specificity and efficacy of the microtubule inhibitor colchicine, valyl colchicine and the legumain-cleavable colchicine prodrug. The prodrug was more toxic to the colorectal cancer HCT116 cells (expressing both the 36kDa active and 56kDa proform of legumain) than SW620 cells (only expressing the 56kDa prolegumain) indicating a relationship between toxicity of the prodrug and activity of legumain in the cells. Also, in monoclonal legumain over-expressing HEK293 cells the prodrug toxicity was higher compared to native HEK293 cells. Furthermore, co-administration of the prodrug either with the potent legumain inhibitor cystatin E/M or the endocytosis inhibitor Dyngo-4a inhibited cell death, indicating that the prodrug toxicity was dependent on both asparaginyl endopeptidase activity and endocytosis. This colchicine prodrug adds to a legumain-activated prodrug strategy approach and could possibly be of use both in targeted anticancer and anti-inflammatory therapy.

Evaluation of P1' substrate specificity of staphylococcal SplB protease.

Staphylococcus aureus is a dangerous human pathogen characterized by growing antibiotic resistance. Virulence of S. aureus relies on a variety of secreted and cell surface associated virulence factors among which certain proteolytic enzymes play an important role. Amid staphylococcal extracellular proteases, those encoded by the spl operon remain poorly characterized, both in terms of enzymology and their physiological role. Initial data demonstrated that Spl proteases exhibit restricted substrate specificity. This study describes development of convenient protein FRET substrates for SplB protease and characterization of the substrate preference of the protease at the P1' position. Kinetic data on hydrolysis of a panel of substrates substituted at the said position is provided.

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from −4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.

Chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose and study of the substrate specificity of HldE

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.

Clostridium Botulinum C3 Exoenzyme: Rho-Inactivating Tool in Cell Biology and a Neurotrophic Agent

C3 exoenzyme from Clostridium botulinum is the prototype of bacterial ADP-ribosyltransferases, which selectively modifies the Rho isoforms RhoA, RhoB and RhoC by covalent attachment of an ADP-ribose moiety. ADP- ribosylation results in inactivation of cellular functions of Rho. Because of its highly restricted substrate specificity, C3 is an established tool in cell biology; to this end C3 is applied as a cell-permeable chimeric toxin. C3 is superior to other molecular biology techniques such as siRNA or knock down approaches as RhoA inactivation or knock down is intrinsically associated with RhoB activation except after C3 treatment. RhoA plays an essential role in axonal growth and repair after neuronal injury. For therapeutic purposes cell- permeable C3 is now locally administered to treat spinal cord injury. Recently, it was reported that ADP-ribosyltransferase activity is not essential for the neurotrophic effect of C3 and that a peptidic fragment of C3 acts neurotrophic.

On the Physiological Role of Cytosolic 5’-nucleotidase II (cN-II): Pathological and Therapeutical Implications.

Among the members of the 5'-nucleotidase family, there is only one membrane-bound ectosolic isoenzyme. This esterase prefers AMP as substrate but can hydrolyze a number of purine and pyrimidine phosphorylated compounds, indicating that no evolutive pressure to develop a more restricted specificity was exerted on this enzyme. On the contrary, five cytosolic isoforms have been evolved, probably by convergent evolution, showing different and restricted substrate specificity. The different isoforms have different level of expression and distribution in organs of vertebrates. The cytosolic nucleotidase specific for IMP and GMP (cN-II), is an enzyme allosterically regulated, structurally strongly conserved and expressed at a low but constant level in all organs and tissues in vertebrates. As far as we know, alteration of cN-II expression is limited to pathological conditions. In this review, we report the results of the modulation of cN-II specific activity exerted by silencing or hyperexpression in different cell types, in the attempt to better understand its role and implications in pathology and therapy.

Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S0-Based Respiration and a Sulfur-Dependent Antioxidant System in Pyrococcus

FAD and NAD(P)H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur-reducing anaerobic hyperthermophiles Pyrococcus horikoshii and Pyrococcus furiosus; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from P. horikoshii to 2.7 Å resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds. These results indicate that the likely in vivo substrates are NAD(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S(8)) in situ is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O(2) and/or H(2)O(2). As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S(0) respiration in Pyrococcus or Thermococcus but appears to participate in oxidative defense in the presence of S(0). When compared to the homologous Npsr enzyme from Shewanella loihica PV-4 and homologous enzymes known to reduce CoA disulfide, the phCoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates.

Label‐free quantitative analysis of the membrane proteome of Bace1 protease knock‐out zebrafish brains

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β-secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter-aided sample preparation, strong-anion exchange fractionation, and label-free quantification. We used bace1-deficient zebrafish and quantified differences in protein levels between wild-type and bace1 -/- zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild-type and bace1 -/- zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty-four membrane proteins accumulated in the bace1 -/- brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican-1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock-out zebrafish tissue and demonstrates that combining gene knock-out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including β-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Surface patterning using scanning electrochemical microscopy to locally trigger a “click” chemistry reaction

We report on the surface micropatterning of conductive surfaces via the electrochemical triggering of a click reaction, the copper(I) catalyzed azide–alkyne cycloaddition reaction (CuAAC) by SECM via a two-step approach: (i) functionalization on the entire surface with azido-aryl groups by using the diazonium approach followed by (ii) the covalent linkage of alkyne-bearing ferrocene by CuAAC within a local area by SECM. More precisely, the click reaction was triggered by Cu(I) catalyst generation for 30min at the SECM tip positioned ≈10μm above the azido-aryl modified surface. The dimension of the spot obtained under these conditions was ≈75μm. The electrochemical imaging by SECM of the ultra thin area locally clicked with ferrocene moieties was made thanks to the electrocatalytic properties of the ferrocene modified surface towards ferrocyanide electrooxidation. This local clicking procedure opens the gate to further controlled functionalization of restricted small substrates.

Profluorogenic Reductase Substrate for Rapid, Selective, and Sensitive Visualization and Detection of Human Cancer Cells that Overexpress NQO1

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability.

Metabolic Downregulation and Inhibition of Carbohydrate Catabolism during Diapause in Embryos ofArtemia franciscana

Diapause embryos were collected from ovigerous females of Artemia franciscana at the Great Salt Lake, Utah, and were synchronized to within 4 h of release. Respiration rate for these freshly released embryos across a subsequent 26-d time course showed a rapid decrease during the first several days followed thereafter by a much slower decline. The overall metabolic depression was estimated to be greater than 99%. However, proton conductance of mitochondria isolated from diapause and postdiapause embryos was identical. Because proton leak is apparently not downregulated during diapause, mitochondrial membrane potential is likely compromised because of the very low metabolic rate observed for diapause embryos. Given that trehalose is the primary fuel used by these embryos, we measured metabolic intermediates along the catabolic pathway from trehalose to acetyl-CoA for both diapause and postdiapause (active) embryos in order to identify sites of metabolic inhibition. Comparison of product-to-substrate ratios for sequential enzymatic steps revealed inhibition during diapause at trehalase, hexokinase, pyruvate kinase, and pyruvate dehydrogenase. Measurements of ATP, ADP, and AMP allowed calculations of substantial decreases in ATP:ADP ratio and in adenylate energy charge during diapause. The phosphorylation of site 1 for pyruvate dehydrogenase (PDH) subunit E1α was higher in diapause embryos than in postdiapause embryos, which is consistent with PDH inhibition during diapause. Taken together, our findings indicate that restricted substrate availability to mitochondria for oxidative phosphorylation contributes to downregulating metabolic rate during diapause.