Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory.

Non-fibrillar soluble oligomeric forms of amyloid-ß peptide (oAß) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAß initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aß, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAß levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAß to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aß on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aß and tau pathology.

Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase.

Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly O-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. We have genetically engineered a series of truncated forms of GA to determine how much of the highly O-glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain. Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes. Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme. Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.

Solvent and viscosity effects on the rate-limiting product release step of glucoamylase during maltose hydrolysis.

Release of product from the active site is the rate-limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoamylase (GA). With GA, an enzymatic conformational change has been associated with the product release step. Solvent characteristics such as viscosity can strongly influence protein conformational changes. Here we show that the rate-limiting step of GA has a rather complex dependence on solvent characteristics. Seven different cosolvents were added to the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low concentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on polymer length of the cosolvent; the longer the polymer, the lower the concentration needed. The maximum increase in catalytic activity at 45 degrees C (40-45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000). A further increase in activity to 60-65% was obtained at 60 degrees C. The linear relationship between ln(kcat) and (viscosity)2 obtained with all the cosolvents provides further evidence that product release is the rate-limiting step in the GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has potential applications for the food industry where high-fructose corn syrup (HFCS) is one of the primary products produced with GA. Since maltodextrin hydrolysis by GA is by far the slowest step in the production of HFCS, increasing the catalytic rate of GA can substantially reduce the process time.

Artificial antibodies for affinity chromatography of homologous proteins: application to blood clotting proteins.

A method to readily isolate antibodies that bind to only one member of a family of homologous proteins is described. A library of different single chain antibody fragments can be displayed on the surface of a bacteriophage vector. Individual antibodies from this library recognizing a particular protein from a family of homologous proteins can be readily isolated by a two-step affinity screening process. In the first step antibodies which bind specifically to the undesired proteins or to homologous regions of the proteins are removed. In the second step, those antibodies specifically recognizing the desired protein are then isolated. Using this procedure and starting with a naive antibody library, a single chain antibody fragment specific to the blood clotting protein, Protein C, which did not recognize either of the homologous proteins, Factor IX or Factor X, was isolated. Similarly an antibody specific to Factor IX, but not Factor X or Protein C, was also isolated. The isolated antibodies can be readily produced, purified, and affixed to sepharose beads for affinity chromatography of the blood clotting factors. One of the key advantages to this procedure over conventional monoclonal antibody isolation is that the antibodies are isolated and produced in vitro so a broad range of related proteins, toxins, viruses, or other products can be targeted.

Specific glycosidase activity isolated from a random phage display antibody library.

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.

Roles of the aromatic side chains in the binding of substrates, inhibitors, and cyclomalto-oligosaccharides to the glucoamylase from Aspergillus niger probed by perturbation difference spectroscopy, chemical modification, and mutagenesis.

The roles of the aromatic side chains of the glucoamylase from Aspergillus niger in the binding of ligands, as determined by difference spectroscopy using four types of inhibitors (a) valienamine-derived, (b) 1-deoxynojirimycins, (c) D-glucono-1,5-lactone, and (d) maltitol, two types of disaccharide substrates (a) alpha-(1----4)-linked and (b) alpha-(1----6)-linked, and three cyclomalto-oligosaccharides (cyclodextrins, CDs) are discussed. An unusual change in absorbance from 300 to 310-320 nm, obtained only with the valienamine-derived inhibitors or when D-glucono-1,5-lactone and maltose are combined, is concluded to arise when subsite 2 is occupied in a transition-state-type of complex. The single mutations of two residues thought to be involved in binding, namely, Tyr116----Ala and Trp120----Phe, alter, but do not abolish this perturbation. The perturbations in the spectra also suggest that maltose and isomaltose have different modes of binding. The following Kd values (M) were determined: acarbose, less than 6 x 10(-12); methyl acarviosinide, 1.6 x 10(-6); and the D-gluco and L-ido forms of hydrogenated acarbose, 1.4 x 10(-8) and 5.2 x 10(-6), respectively. Therefore, both the valienamine moiety and the chain length of acarbose are important for tight binding. In contrast to the valienamine-derived inhibitors, none of the 1-deoxynojirimycin type protected glucoamylase against inactivating oxidation of tryptophanyl residues, although each had a Kd value of approximately 4 x 10(-6) M. There are two distinct carbohydrate-binding areas in glucoamylase, namely, the active site in the catalytic domain and a starch-granule-binding site in the C-terminal domain. The alpha-, beta-, and gamma-CDs have high affinity for the starch-binding domain and low affinity for the active site, whereas the reverse was found for acarbose.

Targeted hydrolysis of Beta-amyloid with engineered antibody fragment.

Accumulation and deposition of beta amyloid (Abeta) play a critical role in the pathogenesis of Alzheimer's Disease (AD), and numerous approaches to control Abeta aggregation are being actively pursued. Brain Abeta levels are controlled by the action of several proteolytic enzymes such as neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin. While up-regulation of these enzymes increased clearance of Abeta in transgenic mouse models of AD, these enzymes have other natural substrates and multiple cleavage sites in Abeta complicating their use for treating AD. Alternatively, immunotherapeutic approaches to clear Abeta are gaining interest. Active and passive immunization studies with Abeta can reduce plaque burden and memory loss, but clinical trials were stopped due to meningioencephalitis in some patients. Naturally occurring proteolytic antibodies have been shown to cleave Abeta, and their serum titers are increased in patients with AD reflecting a protective autoimmune response. These antibodies however cannot cross the blood brain barrier and depend entirely on peripheral clearance to clear Abeta. A potentially non-inflammatory approach to facilitate Abeta clearance and reduce toxicity is to promote hydrolysis of Abeta at its alpha-secretase site using affinity matured single chain antibody fragments (scFvs). Bispecific antibodies consisting of a proteolytic scFv and a targeting scFv can be engineered to selectively supplement and target extracellular alpha-secretase activity and to target toxic Abeta forms facilitating their degradation and clearance without generating an immune response. This strategy represents a suitable paradigm for treating other neurological diseases such as Parkinson's Disease, Lou Gehrig's Disease, and spongiform encephalopathies.

Isolation of an scFv targeting BRG1 using phage display with characterization by AFM.

Remodeling of chromatin is a vitally important event in processes such as transcription and replication. Brahma-related gene 1 (BRG1) protein is the major ATPase subunit in the human Swi/Snf complex (hSwi/Snf), an important example of the family of enzymes that carry out such remodeling events. We have used a recently developed technique, recognition imaging, to better understand the role of BRG1 in remodeling chromatin. In such experiments, a specific antibody against BRG1 is needed. However, we have found that the commercially available polyclonal (CAP) antibodies interact non-specifically with nucleosomes, making it impossible to identify hSwi/Snf (BRG1) in their presence. Here antibody phage display technology is employed for development of an antibody specifically targeting BRG1. The Tomlinson I and J single chain variable fragment (scFv) libraries were used for successful isolation of an anti-BRG1 scFv. We demonstrate that the scFv binds more strongly and with less nonspecific interactions than the CAP antibody. This work lays the groundwork for future studies involving chromatin remodeling.

Application of cross-linked carboxymethyl cellulose degradation by beta-glucosidase and vaginal microbes to toxic shock syndrome.

Eleven bacterial and two yeast strains, four of which were previously identified as having activity on a lightly cross-linked carboxymethyl cellulose (CLD-2) found in one type of superabsorbent tampon, were grown on a variety of substrates, most containing cellulosics. None produced detectable amounts of cellulases, but all elaborated beta-glucosidase. None of these 13 strains nor 3 commercially obtained beta-glucosidase preparations could hydrolyze CLD-2, although a commercial cellulase and two other bacterial preparations known to produce cellulases could. Based on these results, it appears that previous work suggesting that the degradation of CLD-2 by vaginal microbes and beta-glucosidase is implicated in the production by Staphylococcus aureus of toxin causing toxic shock syndrome must be reevaluated.

Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes.

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose from non-reducing ends of starch with an approximately 300-fold (kcat/Km) preference for the alpha-1,4- over the alpha-1,6-glucosidic linkage determined using the substrates maltose and isomaltose. It is postulated that as most amylolytic enzymes act on either the alpha-1,4- or alpha-1,6-linkages, sequence comparison between active-site regions should enable the correlation of the substrate bond specificity with particular residues at key positions. Therefore, the already high bond-type selectivity in Aspergillus glucoamylase could theoretically be augmented further by three single mutations, Ser119-->Tyr, Gly183-->Lys and Ser184-->His, in two separate active-site regions. These mutants all had slight increases in activity as compared with the wild-type enzyme towards the alpha-1,4-linked maltose; this was due to lower Km values as well as small decreases in activity towards isomaltose. This latter decrease in activity was a result of higher Km values and a decrease in kcat for the Ser184-->His mutant. As a consequence, the selectivity of the three glucoamylase mutants for alpha-1,4- over alpha-1,6-linked disaccharides is enhanced 2.3- to 3.5-fold. In addition, the introduction of a cationic side chain in Gly183-->Lys and Ser184-->His glucoamylase, broadens the optimal pH range for activity towards acidic as well as alkaline conditions.

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex.

Molecular recognition and site-directed mutagenesis are used in combination to identify kinetically, transition state interactions between glucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180----Gln GA had indicated a role in substrate binding for Glu180 (Sierks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193-198). Here, changes in activation energies calculated from measured kcat/Km values for a series of deoxygenated maltose analogues indicate hydrogen bonding between the mutant enzyme and the 3-OH group of the reducing end sugar ring. Using the same substrate analogues and determining activation energies with wild-type GA an additional hydrogen bond with the 2-OH group of maltose is attributed to an interaction with the carboxylate Glu180. This novel combination of molecular recognition and site-directed mutagenesis enables an enzyme substrate transition state contact to be identified and characterized even without access to the three dimensional structure of the enzyme. Given the distant structural relationships between glucoamylases and several starch hydrolases (Svensson,B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activity of these related enzymes.

Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis.

Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.

Identification of enzyme-substrate and enzyme-product complexes in the catalytic mechanism of glucoamylase from Aspergillus awamori.

Intermediates in the catalytic mechanism of Aspergillus awamori glucoamylase (GA) were identified by studying pre-steady-state and steady-state kinetics of the wild-type GA/maltose and Trp120 -->Phe GA/maltotriose reactions in H2O and D2O. Pre-steady-state fluorescence signal analysis was carried out to ascertain the relative intrinsic fluorescence of the enzyme intermediates. A three-step minimal pathway for oligosaccharide hydrolysis represented by E + Gx (k1) reversible (k-1) EGX (k2)reversible(k-2) EP (kcat)--> E + P is proposed. The first step, represented by the association constant K1 (k1/k-1), depicts the fast formation of enzyme-substrate complex and is the primary factor in fluorescence quenching. A 2.7-fold increase in K1 with D2O as solvent is observed with both enzymes due to the cumulative effect of deuterium on complex hydrogen bonding at the active site. The second step further quenches the enzyme fluorescence and is identified as the hydrolytic step, forming an enzyme-product complex. Both k2 and k-2 values show similar 2-fold decreases in D2O for both enzymes, consistent with the microscopic reversibility of the hydrolytic reaction. The solvent isotopic effect on the hydrolytic step is likely due to either abstraction of an exchangeable proton from the general acid Glu179 or directed addition of water to the oxocarbonium ion intermediate by the general base Glu400. No significant isotope effect was observed on the steady-state kcat value for wild-type GA with maltose, indicating a ronhydrolytic step as rate-limiting. The third step, a posthydrolytic rate-determining step, is the product release as evident from steady-state kinetics with wild-type and Trp120-->Phe GAs using alpha-D-glucosyl fluoride.

Minimizing nonproductive substrate binding: a new look at glucoamylase subsite affinities.

A subsite model as proposed by Hiromi [Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6] has been applied to various hydrolases including glucoamylase (GA). The model assumes a single enzyme complex, a hydrolytic rate constant which is independent of substrate length, and a ratelimiting hydrolytic step. Recent kinetic studies with GA contradict these assumptions. Here we reevaluate the substrate binding of GA studying the pre-steady-state kinetics with glucose, which is reported here for the first time, and maltose. The association equilibrium constants for glucose and maltose interactions with wild-type and Trp120-->Phe GA from Aspergillus awamori in H2O and D2O buffers were obtained. Kinetic results indicate that a single glucose molecule binds to GA weakly by a single-step mechanism, E + G1<-->EG1, under the conditions studied. Similar fluorescence intensities of the GA-glucose and GA-maltose complexes, the high tryptophan concentration around subsite 1, crystal structures of various inhibitor complexes, pre-steady-state and steady-state modeling, feasibility of condensation reactions, and other evidence strongly suggest that glucose binds at subsite 1. These results conflict with the high subsite 2 and low subsite 1 affinities obtained using Hiromi's model. Using the substrate association constants for glucose and maltose obtained by pre-steady-state kinetics, the affinity of alpha-glucose for subsite 1 is shown to be substantially higher than the apparent affinity of glucose for subsite 2. We propose a GA catalytic mechanism whereby substrate binding is initiated by subsite 1 interactions with the nonreducing end of the oligosaccharide substrate, minimizing nonproductive substrate binding. Through conformational changes, entropic contributions, and increased local concentration, subsite 2 subsequently has enhanced affinity for the second covalently linked glucosyl residue.

Functional and structural roles of the highly conserved Trp120 loop region of glucoamylase from Aspergillus awamori.

The functional role of a loop region, highly conserved among glucoamylase and other starch hydrolases which also includes the essential Trp120 of Aspergillus awamori, is investigated. Residues 121-125 of A. awamori glucoamylase were singly substituted, and their individual effects on catalytic activity and thermal stability were determined. The Arg122-->Tyr mutation displayed opposing effects for shorter and longer maltooligosaccharide substrates, K(m) decreasing for shorter substrates but increasing for longer substrates. The Pro123-->Gly mutation decreases the thermal stability of glucoamylase by 19 degrees C with little effect on activity. The Gln124-->His substitution decreases k(cat) for all substrates 10-15-fold. Gly121-->Thr and Arg125-->Lys had only minor effects on glucoamylase activity. While Arg122-->Tyr, Gln124-->His, and the previously constructed Trp120-->Phe [Sierks, M. R., Svensson, B., Ford, C., & Reilly, P. J. (1989) Protein Eng. 2, 621-625] glucoamylases have significantly reduced activity toward maltose hydrolysis, all mutations in the Trp120 loop region retain wild-type level activity toward alpha-D-glucosyl fluoride hydrolysis. The Trp120 loop region therefore plays a major role in directing conformational changes controlling the postulated rate-limiting product release step, even though only Trp120 is indicated to interact with acarbose in the crystal structure [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639]. Side chains of residues 116, 120, 122, and 124 oriented in one direction play crucial roles in the enzyme mechanism, while side chains of residues 119, 121, 123, and 125, oriented in the opposite direction, play only minor roles.

Catalytic mechanism of glucoamylase probed by mutagenesis in conjunction with hydrolysis of alpha-D-glucopyranosyl fluoride and maltooligosaccharides.

The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using alpha-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116-->Ala, Trp120-->Phe, Asp176-->Asn, and Glu400-->Gln. These replacements decreased the activity (kcat/KM) toward maltose by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GAs, showed wild-type and twice wild-type level activity, while Asp176-->Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild-type GA complexed with GF, the pH-activity (kcat) profile shows a pKa of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis.

Functional roles of the invariant aspartic acid 55, tyrosine 306, and aspartic acid 309 in glucoamylase from Aspergillus awamori studied by mutagenesis.

Three mutants, Asp55-Gly, Tyr306-->Phe, and Asp309-->Asn, of Aspergillus awamori glucoamylase (identical to Aspergillus niger glucoamylase) were constructed to elucidate the roles of two conserved regions within fungal glucoamylases. Kinetic studies indicate that both of these regions are closely associated with activity. The Asp55-->Gly mutation decreases the kcat approximately 200 times toward maltose and isomaltose, while KM values remain similar to the wild-type. This localizes Asp55 to subsite 1 of glucoamylase where it affects catalytic activity, but not ground-state binding. The pKa value of the catalytic general acid, Glu179, is 1 pH unit lower in that mutant compared to wild-type enzyme, confirming the proximity of Asp55 to the site of catalysis. Tyr306-->Phe is highly active, but affects binding in subsite 2. It moreover shows enhanced binding in the fourth subsite, suggesting that the conserved region around residue 306 interacts with Trp120, a critical residue that directs conformational changes stabilizing the transition-state structure. Finally, the Asp309-->Asn mutation decreases the kcat for isomaltose hydrolysis around 200-fold, but only 30-fold for maltose. This specific effect on the hydrolysis of the alpha-1,6-linked substrate locates Asp309 to subsite 2. Substitution of Asp309 influences affinities of distant subsites, especially subsite 4, similar to mutations of other carboxylic acid residues situated near subsites 1 and 2.

Reaction mechanisms of Trp120-->Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose.

Interactions of wild-type and Trp120-->Phe glucoamylase with maltooligodextrin (Gx) substrates and the tight-binding inhibitor acarbose (A) were investigated here using stopped-flow fluorescence spectroscopy and steady-state kinetic measurements. All wild-type and Trp120-->Phe glucoamylase reactions followed the three-step model E + Gx(or A) (k1) <==> (k-1) EGx (or A) (k2) <==> (k-2) E*Gx(or A) (k3) --> E + P or E-A, previously shown to account for the glucoamylase-maltose system [Olsen, K., Svensson, B., & Christensen, U. (1992) Eur. J. Biochem. 209, 777-784]. K1 = k-1/k1, k2, and k-2, and the catalytic constant, k3, are determined. Binding of maltooligodextrins in the first reaction step is weak, with little difference between wild-type and Trp120-->Phe glucoamylase. The second step, involving a conformational change, in contrast, is strongly influenced by the mutation and by the substrate length. Here wild-type glucoamylase reacts faster and forms more stable intermediates the longer the substrate. In contrast, Trp120-->Phe reacts slower the longer the substrate. The effect of the mutation is thus smallest on maltose. The Trp120-->Phe substitution reduces the fluorescence signal only by 12-20%, indicating that other tryptophanyl residues are important in reporting the conformational change. Trp120 also strongly influences the actual catalytic step, since the mutation decreases the kc values 30-80-fold. Acarbose behaves similar to maltotetraose in the first and the second steps with wild-type but not the Trp120-->Phe glucoamylase. Also, a third step in the acarbose reaction which parallels the catalytic step is strongly affected by the mutation. The rate constant k3 increases 200-fold.

Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates.

The specificity constants, kcat/KM, were determined for glucose oxidase and glucose dehydrogenase using deoxy-D-glucose derivatives and for glucoamylase using deoxy-D-maltose derivatives as substrates. Transition-state interactions between the substrate intermediates and the enzymes were characterized by the observed kcat/Km values and found to be very similar. The binding energy contributions of individual sugar hydroxyl groups in the enzyme/substrate complexes were calculated using the relationship delta(delta G) = -RT ln [(kcat/KM)deoxy/(kcat/KM)hydroxyl] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4- and 6-OH groups (4'- and 6'-OH in maltose), where changes in binding energies from 10 to 18 kJ/mol suggested strong hydrogen bonds between the enzymes and these substrate OH groups. The 3-OH (3'-OH in maltose) was involved in weaker interactions, while the 2-OH (2'-OH in maltose) had a very small if any role in transition-state binding. The three enzyme-substrate transition-state interactions were compared using linear free energy relationships (Withers, S. G., & Rupitz, K. (1990) Biochemistry 29, 6405-6409) in which the set of kcat/KM values obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (rho) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology.(ABSTRACT TRUNCATED AT 250 WORDS)

Starch- and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (beta/alpha)8-barrel domain and evolutionary relationship to other amylolytic enzymes.

Sequence alignment and structure prediction are used to locate catalytic alpha-amylase-type (beta/alpha)8-barrel domains and the positions of their beta-strands and alpha-helices in isoamylase, pullulanase, neopullulanase, alpha-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes--all enzymes involved in hydrolysis or synthesis of alpha-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of beta-->alpha loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved beta-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.