Dynamical study,hydrogen bond analysis,and constant pH simulations of the beta carbonic anhydrase of Methanobacterium thermoautotrophicum

Within the five classes (α, β, γ, δ, and ζ) of carbonic anhydrases (CAs) the first two, containing mammal and plant representatives, are the most studied among all CAs. In this study, we have focused our investigation on the beta-class carbonic anhydrase of Methanobacterium thermoautotrophicum. We investigated both the importance of the Asp-Arg dyad near the catalytic zinc-bound water and the possible roles that water molecules within the active site and residues near the entrance of the catalytic cleft have on the first step of the enzyme’s reaction mechanism. Hydrogen-bonding analysis of selected residues within the active site and constant pH replica exchange molecular dynamics constant pH replica exchange simulations were performed. The latter was done in order to evaluate the pK_a values of possible proton acceptors. We found an intricate hydrogen-bonding network involving two acidic residues within the active site, Asp16 and Asp34, and the catalytic water molecule. We also observed a very strong interaction between the zinc-bound water and residues Asp34 and Arg36. This interaction was not significantly affected by the change in the protonation state of both the catalytic water and aspartate residue 34. The pK_a analysis show that the effect of the R36A mutation affects not only the possible proton acceptors, but also the catalytic water itself.     

Enzyme activity during the metabolism of glycogen

Summary Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii.After incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO₄ (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO₄, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

In silico and in vivo analyses reveal key metabolic pathways enabling the fermentative utilization of glycerol in Escherichia coli

Most microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H₂, complementing the previously reported role of 1,2-propanediol synthesis under acidic conditions. In this new redox balancing mode, H₂ evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol-3-phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase-dependent H₂ evolution. Despite the importance of the GK-G3PDH route under alkaline conditions, we found that the NADH-generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)-dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system-wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA-DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose-1,6-bisphosphatases and the fructose phosphate bypass.     

The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Str?hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.     

Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024

A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0–7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4–10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent K _m values for validoxylamine A and d-glucose were 8.3 and 1.1 mM, respectively. Cu²⁺, Ag²⁺, and Hg₂Cl₂ inhibited the activity of G3DH.     

A tale of two plasticities: leaf hydraulic conductances and related traits diverge for two tropical epiphytes from contrasting light environments

We compared the effects of different light environments on leaf hydraulic conductance (K_leaf) for two congeneric epiphytes, the tank bromeliads Guzmania lingulata (L.) Mez and Guzmania monostachia (L.) Rusby ex Mez. They occur sympatrically at the study site, although G. monostachia is both wider ranging and typically found in higher light. We collected plants from two levels of irradiance and measured K_leaf as well as related morphological and anatomical traits. Leaf xylem conductance (K_xy) was estimated from tracheid dimensions, and leaf conductance outside the xylem (K_ox) was derived from a leaky cable model. For G. monostachia, but not for G. lingulata, K_leaf and K_xy were significantly higher in high light conditions. Under both light conditions, K_xy and K_ox were co‐limiting for the two species, and all conductances were in the low range for angiosperms. With respect to hydraulic conductances and a number of related anatomical traits, G. monostachia exhibited greater plasticity than did G. lingulata, which responded to high light chiefly by reducing leaf size. The positive plasticity of leaf hydraulic traits in varying light environments in G. monostachia contrasted with negative plasticity in leaf size for G. lingulata, suggesting that G. monostachia may be better able to respond to forest conditions that are likely to be warmer and more disturbed in the future.     

Highly conserved glycine 86 and arginine 87 residues contribute differently to the structure and activity of the mature HIV‐1 protease

The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR_G86A and PR_G86S). While PR_G86S had undetectable catalytic activity, PR_G86A exhibited ～6000‐fold lower catalytic activity than PR. ¹H‐¹⁵N NMR correlation spectra revealed that PR_G86A and PR_G86S are dimeric, exhibiting dimer dissociation constants (K_d) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (K_d > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR_G86A and PR_G86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Activities and properties of turbellarian and cestode glucose-6-phosphatases

There were studied activities and properties of mitochondrial and microsomal glucose6-phosphatases (G6Pases) in the free-living turbellarian Phagocata sibirica and cestode Bothriocephalus scorpii. Action of various effectors (sodium fluoride, glucose, HCO₃, citrate, Cu²⁺, DTT, EDTA, ATP, ADP) on the enzyme activity was studied. The obtained results and literature data demonstrate that G6Pase is present in various muscles of different representatives of the animal kingdom. The conclusion could be made that invertebrate G6Pase releases glucose from glycogen and gluconeogenic precursors.     

Benzotriazole-Based Polymer Acceptor for High-Efficiency All-Polymer Solar Cells with High Photocurrent and Low Voltage Loss

The power conversion efficiencies (PCEs) of all-polymer solar cells (all-PSCs) have already exceeded 17%. However, the limited absorption range of an all-polymer system results in significantly reduced short-circuit current density (J_sc), which eventually influences the PCE improvement. To broaden the light absorption of polymer acceptors, herein, benzotriazole is introduced in the core unit of small molecule acceptors and thus two narrow-bandgap polymer acceptors named PTz-BO and PTz-C11 featuring the same molecular backbone and different side-chain length are synthesized. Compared with PTz-C11, the PTz-BO based-all PSCs deliver a slightly reduced J_sc, a large open-circuit voltage (V_oc) and a low voltage loss below 0.50 V. Moreover, ternary all-PSCs are constructed by introducing PTz-C11 as a guest component. Benefiting from the reduced recombination, improved exciton generation and dissociation, and balanced charge transport, a high efficiency of 16.58% is obtained for the ternary all-PSCs, with a high J_sc over 25 mA cm⁻² without sacrificing the V_oc. Such result represents the highest efficiency reported for benzotriazole-based all-PSCs in the literature thus far. This work demonstrates the great potential of benzotriazole for the synthesis of efficient narrow-bandgap polymer acceptors.     

Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor

The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5 mM).     

Modeling of ligand binding to G protein coupled receptors: cannabinoid CB1, CB2 and adrenergic β2AR

Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) G protein coupled receptors. Docking of agonists and antagonists to CB₁ and CB₂ cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch and its possible participation in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB₁ and CB₂ receptor models were constructed based on the adenosine A_2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β₂AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.     

A novel enzymatic dehalogenation of (R)-3-chloro-1,2-propanediol inAlcaligenes sp. DS-S-7G

The mechanism of the novel enzymatic dehalogenating action of (R)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was investigated. The (R)-MCH-dechlorinating enzyme system ofAlcaligenes sp. DS-S-7G, which stereoselectively assimilated (R)-MCH from the racemate, was composed of two components (Enzyme 1 and Enzyme 2). Enzyme 1 was a flavoprotein with a relative molecular mass (M _r) of 70,000 and was composed of two kinds of polypeptides (58,000 and 16,000). The enzyme exhibited activity for converting (R)-MCH to hydroxyacetone with the liberation of chloride ions under aerobic conditions. On the other hand, Enzyme 2 with anM _r of 86,000, which was also composed of two kinds of polypeptides (33,000 and 53,000), showed no dechlorinating activity for (R)-MCH. However, in the presence of NAD⁺, when Enzyme 1 was conjugated with Enzyme 2 in the (R)-MCH-dechlorinating reaction, the co-operative dechlorinating activity was four to five times higher than that by Enzyme 1 alone. (R)-MCH was finally degraded to acetic acid and formic acid by the joint action of the two enzymes. These facts indicate that (R)-MCH is oxidatively dechlorinated by the two enzymes in the presence of NAD⁺ inAlcaligenes sp. DS-S-7G.     

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q_o site). We identified the G37V change within the cytochrome b Q_i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q_o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.     

Variation in the degree of branching of the glycogens and an approximate relationship between average exterior chain length and iodine staining

Precise evaluation of the branching characteristics of glycogen complex from a variety of sources was made by replicate determinations of end groups by a reproducible periodate oxidation method and by replicate determinations of the percentage degradation by exhaustive β-amylase activity. Also, the iodine staining characteristics of these samples were studied. The following observations were made. During the synthesis of glycogen by baker's yeast, there was a significant increase in the average chain length of the glycogen, and the maximal optical absorbance A_M of the glycogen–iodine complex doubled. The A_M decreased linearly with increasing temperature, and increases in iodine concentration caused increases in both the A_M and the wavelength of maximal absorbance. There was a general increase in the A_M of the glycogen with increasing exterior chain length, but there were minor deviations due to some dependence on the source of the glycogen. Unlike the linear relationship between the A_M per chain and the average chain length of the amyloses, the A_M per chain was exponentially related to the average exterior chain length of the glycogens.     

So close,so different: geothermal flux shapes divergent soil microbial communities at neighbouring sites

This study is focused on the (micro)biogeochemical features of two close geothermal sites (FAV1 and FAV2), both selected at the main exhalative area of Pantelleria Island, Italy. A previous biogeochemical survey revealed high CH₄ consumption and the presence of a diverse community of methanotrophs at FAV2 site, whereas the close site FAV1 was apparently devoid of methanotrophs and recorded no CH₄ consumption. Next‐Generation Sequencing (NGS) techniques were applied to describe the bacterial and archaeal communities which have been linked to the physicochemical conditions and the geothermal sources of energy available at the two sites. Both sites are dominated by Bacteria and host a negligible component of ammonia‐oxidizing Archaea (phylum Thaumarchaeota). The FAV2 bacterial community is characterized by an extraordinary diversity of methanotrophs, with 40% of the sequences assigned to Methylocaldum, Methylobacter (Gammaproteobacteria) and Bejerickia (Alphaproteobacteria); conversely, a community of thermo‐acidophilic chemolithotrophs (Acidithiobacillus, Nitrosococcus) or putative chemolithotrophs (Ktedonobacter) dominates the FAV1 community, in the absence of methanotrophs. Since physical andchemical factors of FAV1, such as temperature and pH, cannot be considered limiting for methanotrophy, it is hypothesized that the main limiting factor for methanotrophs could be high NH₄⁺ concentration. At the same time, abundant availability of NH₄⁺ and other high energy electron donors and acceptors determined by the hydrothermal flux in this site create more energetically favourable conditions for chemolithotrophs that outcompete methanotrophs in non‐nitrogen‐limited soils.     

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).     

Molecular Shape and Packing in Crystals of Soybean β-Amylase

The structure of soybean β-amylase in trigonal (P3₂21) crystals was determined at 4.5 Å resolution by X-ray crystallographic techniques using the isomorphous replacement method. X-Ray diffraction data were collected by the screened precession method for the native enzyme and two heavy atom derivatives. The shape of the enzyme molecule and the locations of mercurial binding are presented. The molecule appeared to be composed of two domains: the larger domain contains one mercurial site on its surface and the smaller domain has another mercurial site, which seemed to be the so-called essential sulfhydryl group. A distinct cleft formed between the domains near the latter sulfhydryl group may be a substrate binding region.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity