Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit

Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes.     

Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant

Production of a raw starch-digesting glucoamylase O (GA O) by protease-negative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA O was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and pH stability, but it was different in molecular weight and carbohydrate content (250,000 and 24.3% for GA O, 90,000 and ca. 7% for GA I, respectively). The chitin-bound GA O hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA O with subtilisin led to the formation of a modified GA O (molecular weight, 170,000), which hydrolyzed glycogen 100%, similar to GA O and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,000) derived by treatment with substilisin hydrolyzed glycogen up to only 80% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimide-oxidized GA O lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Actions of glycogen synthase and prosphorylase of rabbit-skeletal muscle on modified glycogens

The high reactivities exhibited by rabbit-muscle synthase and phosphorylase for unmodified glycogen-acceptors decrease progressively, presumably because of a large increase in apparent K_m as the glycogen molecule is converted into its component maltosaccharide chains by the debranching enzyme, isoamylase. Elongation of the outer chains of glycogen acceptor also results in decreased reactivities of the two transglucosylases and this is shown, for phosphorylase acting in the direction of glucan synthesis, to be caused by a decrease in the V_max of the reaction. A partial restoration of the degradative reactivity of phosphorylase by a limited alpha-amylolysis of the long outer-chains of modified glycogen suggests a role of cytoplasmic alpha-amylase in mammalian glycogen metabolism.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.     

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.     

Evidence for the proximity of a cysteinyl and a tyrosyl residue in the active site of 6-phosphogluconate dehydrogenase

The treatment of 6-phosphogluconate dehydrogenase from Candida utilis with dansyl chloride causes the modification of one amino acid residue per enzyme subunit and the inactivation of the enzyme. Either a cysteine or a tyrosine residue can be modified, depending on the pH of the reaction mixture. The dansyl residue can be transferred from one residue to the other suggesting that the two amino acid residues are close in the tridimensional structure of the active site of the enzyme.     

Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.     

Cysteine residues at the active site of glutamine synthetase from spinach leaves

Titration of cysteine residues of spinach glutamine synthetase with 5-5' dithiobis (2-nitrobenzoic acid) indicates that there are five such residues per monomer of enzyme and that two of these five are on the surface of the molecule. The presence of substrates, or either of the competitive inhibitors methionine sulfoximine or phosphinothricin, completely protects both of the surface sulfhydryls from titration. This suggests that both are located at the active site. In the absence of Mg2+ and ATP, both surface sulfhydryls must be modified before loss of activity. We conclude that while both of the cysteine residues are located at the active site, only one of them may be involved in catalysis. Because the cysteine residue which is implicated in catalysis can be protected by Mg2+ and ATP, we believe that it may be located at or near the binding site of these ligands.     

Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is modified and inhibited by p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The modification appears to occur at the NAD(H)-binding site when TH alone or TH in the presence of NADPH is incubated with FSBA. However, when this site is protected by NADH, then FSBA inhibits TH more slowly and modifies a different, though specific, site. This second site could be the NADP(H)-binding site. Using [3H]FSBA in the presence of NADPH, the NAD(H)-binding site was modified, and a single tryptic peptide carrying the label was isolated and sequenced. The amino acid sequence of this peptide was Glu-Ser-Gly-Glu-Gly-Gln-Gly-Gly-Tyr*-Ala-Lys. The modified residue was Tyr. The labeled peptide isolated after incubating TH with [3H]FSBA in the presence of NADH could not be completely purified. However, amino acid analysis and partial sequencing made it possible to identify this segment on the amino acid sequence of bovine TH as derived from its cDNA by Yamaguchi et al. (private communication).     

Proposals for the catalytic mechanism of glycogen phosphorylase b prompted by crystallographic studies on glucose 1-phosphate binding

The crystal structure of glycogen phosphorylase b in the presence of the weak activator 2 mm-inosine 5′-phosphate has been solved at 3 Å resolution. The binding interactions of the substrate, glucose 1-phosphate, at the catalytic site are described. The nearby presence (6 Å) of the essential co-factor, pyridoxal phosphate, is consistent with biochemical studies but an analysis of the way in which this group might act in catalysis leads to results that are inconsistent with solution studies. Moreover it is difficult to accommodate a glycogen substrate with its terminal glucose in the position defined by glucose 1-phosphate. Model-building studies show that an alternative binding mode for glucose 1-phosphate is possible and that this alternative mode allows a glycogen substrate to be fitted with ease. The alternative binding site leads directly to proposals for the mechanism in which the phosphate group of pyridoxal phosphate acts as a nucleophile and the imidazole of histidine 376 functions as a general acid. It is suggested that these are the essential features of the catalytic mechanism and that, in the absence of the second substrate, glycogen, and in the absence of AMP, the enzyme binds glucose 1-phosphate in a non-productive mode. Conversion of the enzyme to the active conformation through association with AMP may result in conformational changes that direct the binding to the productive mode.     

A simple two-step approach for introducing a protected diaminedithiol chelator during solid-phase assembly of peptides

A simple synthetic strategy is described to incorporate a protected diaminedithiol (N(2)S(2)) chelator during Fmoc solid-phase synthesis of short peptides. The resulting constructs could be efficiently labeled with technetium-99m (99mTc). The chelator was assembled at the N-terminus of peptides in a two-step procedure where the deprotected terminal amino group was first reacted with di-Fmoc-diaminopropionic acid (Fmoc-DAP-[Fmoc]-OH). The two protected amino groups were then simultaneously deprotected and subsequently reacted with S-benzoylthiolglycolic acid (TGA) to generate a protected N(2)S(2) chelator. This metal binding site was introduced into di- and tripeptides. Each peptide construct was composed of a C-terminal lysine residue and an N-terminal diaminopropionic moiety modified to create the chelator site. The epsilon-amino group at the C-terminal lysine was further derivatized with a nitroimidazole group to facilitate cellular retention. The resulting constructs were then cleaved from the resin support, purified, and labeled with [99mTc]pertechnetate. Six constructs were prepared differing by a single amino acid inserted between the diaminopropionic acid and lysine residues. Optimal labeling yields of >70% were achieved around neutral pH and heating at 75 degrees C for 10 min. Purified 99mTc-labeled constructs were found to accumulate in Chinese hamster ovary (CHO) cells in vitro as a function of charge and hydrophobicity.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.     

Phosphorylation of troponin and myosin light chain by cAMP-independent casein kinase-2 from rabbit skeletal muscle

Casein kinase-2 from rabbit skeletal muscle was found to phosphorylate, in addition to glycogen synthase, troponin from skeletal muscle, and myosin light chain from smooth muscle. Troponin T and the 20,000 M_r myosin light chain are phosphorylated by casein kinase-2 at much greater rates than glycogen synthase. The V values for the phosphorylation of troponin and myosin light chain are nearly an order of magnitude greater than that of glycogen synthase; however, the K_m values for these two substrates are greater than that for glycogen synthase. The kinase activities with the various protein substrates are stimulated approximately three- and fivefold by 5 mm spermidine and 3 mm spermine, respectively. Heparin is a potent inhibitor of the kinase when casein, glycogen synthase, or myosin light chain is the substrate. However, with troponin as substrate the kinase is relatively insensitive to inhibition by heparin. The amount of heparin required for 50% inhibition with troponin as substrate is at least 10 times greater than with casein as substrate. The phosphorylation of troponin by casein kinase-2 results in the incorporation of phosphate into two major tryptic peptides, which are different from those phosphorylated by casein kinase-1. The site in myosin light chain phosphorylated by casein kinase-2 is different from that phosphorylated by myosin light chain kinase.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

The structure of glycogen phosphorylase alpha at 2.5 A resolution

The structure of the glucose-inhibited form of glycogen phosphorylase a has been determined at a resolution of 2.5 Å. With the aid of the primary sequence derived by Titani et al. (1977) for this enzyme, we have constructed an atomic model of the 97,400 molecular weight monomer. A substantial improvement in the electron density map over that reported previously (Fletterick et al., 1976b) was achieved by extension of the data set to 2.5 Å and the inclusion of three additional “heavy-atom” derivatives in the phasing procedure. Main-chain and side-chain electron density are clearly resolved in the map, allowing an unambiguous correlation with the published primary structure. The course of the polypeptide backbone in the C-terminal half of the molecule has been modified at two positions from that reported in the 3.0 Å resolution interpretation.The enzyme is clearly organized into two domains, both with $\text{α}\text{β}$ packing topology. The catalytic site lies in a crevice at the interface between the two domains. α-d-Glucose, which stabilizes the inactive (T) conformation in the parent crystal, is bound at this site in the C_(6′) chair equatorial conformation within 6 Å of the pyridoxal phosphate coenzyme which is covalently bound through the ?-amino group of lysine 679.The larger, N-terminal domain is differentiated by folding architecture and tertiary contacts into two lobes or subdomains which share the same β-sheet backbone: the predominantly helical glycogen storage (maltoheptaose binding) lobe and the N-terminal subdomain. The latter is involved in a variety of protein-protein interactions with the monomer related by the 2-fold axis of the physiological dimer, and contains the serine 14-phosphate moiety and the AMP (positive effector) binding site. The core of the second domain is the complex (βαβ)′ folding unit previously characterized as the nucleotide binding fold (Rao &; Rossmann, 1973).