Subsite Structure Research Articles

Subsite structure and mode of action of native and modified .ALPHA.-amylase from porcine pancreas.

The subsite structure of an active component of porcine pancreatic α-amylase, called PPA II, and modified PPA II (M2) obtained with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), were estimated from their mode of action and rate parameters of hydrolyses of maltooligosaccharides. These results indicated that the native and modified PPA II have five subsites with the catalytic site located between the third and fourth subsites from the nonreducing end. The subsite structure calculated for M2 differed from that of PPA II; subsite affinities of PPA II were calculated to be 1.6, 3.9 and 2.8 kcal/mol for subsites 1, 2 and 5, respectively, whereas corresponding values for M2 were 1.5, 1.4 and 2.6 kcal/mol, respectively. The sum of affinities of the third and fourth subsites was calculated to be –3.6 kcal/mol for PPA II and –2.1 kcal/mol for M2, respectively. Evidently the mode of action of PPA II was changed by the introduction of bulky TNB- ion at or near subsite 2. The difference of the mode of action between PP...

The subsite structures of guanine-specific ribonucleases and a guanine-preferential ribonuclease. Cleavage of oligoinosinic acids and poly I.

In order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guanine-preferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI greater than p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n = 2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.

Ultraviolet difference spectroscopic analysis of the saccharide-binding properties of Ricinus communis agglutinin

The nature of the binding of saccharides to Ricinus communis agglutinin was studied by ultraviolet difference spectroscopy. Upon binding of galactose and galactose-containing saccharides, R. communis agglutinin displayed difference spectra with an extreme maximum at 291–293 nm and a smaller maximum at 284–285 nm. Such difference spectra suggest that the environment of a tryptophan residue located at or near the saccharide-binding site of R. communis agglutinin is being changed by an interaction between a tryptophan residue and the bound saccharides. The value of the difference spectra (Δε) increased upon progressive addition of saccharide until the saccharide binding site was saturated with ligand. From the increase in Δε at 291–293 nm, the association constants were obtained for the R. communis agglutinin-saccharide interaction over the temperature range 5–35°C and various pH values. The results clearly demonstrate that the association constants are nearly equal in the range of pH 5–8, but decrease beyond the above pH range and with elevation of temperature. From the thermodynamic parameters for the binding of various saccharides to R. communis agglutinin, we suggest that there exists a subsite structure in the saccharide-binding site of the R. communis agglutinin molecule.

アミラーゼ反応に関するサブサイト理論とその展開

The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. The subsite structures of several exo- and endo-amylases and an α-glucosidase were shown in histograms, from which the binding modes of linear substrates and glucose can be predicted.The theory is effectively utilized as follows: 1) Interpretation of the substrate specificity of the amylase. 2) Calculation of the change in product distribution in the course of hydrolytic reaction catalyzed by the amylase. 3) Characterization of the nature of subsites by spectroscopic methods and chemical modification. 4) Prediction of change in action pattern by chemical modification. 5) Detailed analysis of mechanisms of amylase-catalyzed reactions by fast reaction techniques.Several actual examples of application of the theory to amylases, including glucoamylase, Taka-amylase A and bacterial liquefying α-amylase, were described.

Subsite structure and action mode of the .ALPHA.-amylase from Thermoactinomyces vulgaris.

The subsite structure of Thermoactinomyces vulgaris α-amylase was estimated from its action mode and rate parameters of hydrolysis on maltooligosaccharides. These results led to the conclusion that this α-amylase has six subsites with the catalytic site located between the third and fourth subsites from the non-reducing end side. Subsite affinities were calculated to be 0.38, 5.46, 2.72 and 0.23 kcal/mol for subsites 1, 2, 5 and 6, respectively, and the sum of the affinities of subsite 3 and 4 to be - 3.41 kcal/mol. The unique action mode of this α-amylase on various substrates was interpreted in terms of the subsite structure.

Some evidence suggesting the existence of P2 and B3 sites in the active site of bovine pancreatic ribonuclease A.

In order to clarify the subsite structure of ribonuclease A (RNase A), the interactions of pdTp, pAp, dTpdAp, and pdTpdAp with RNase A were investigated by means of kinetic studies and 31P NMR spectroscopy. The pH profile of the 31P NMR spectrum of RNase A-pdTp complex indicated the interaction of the 3'- and 5'-phosphates with RNase A. The signal of 3'-phosphate of pdTp in the presence of RNase A gave a characteristic titration curve indicating the participation of more than 2 ionic groups in the P1 subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indicated that pAp interacted with RNase A at the P1, B2, and P2 sites. dTpdAp and pdTpdAp inhibited RNase A action more markedly than dTpdA, indicating the contribution of 3'- and 5'-terminal phosphate groups attached to dTpdA to the affinity of RNase A. The 31P NMR spectra of RNase A-dTpdAp and pdTpdAp complexes excluded the possible interaction of the monoester type phosphate of the inhibitors with the P1 site of RNase A, thus indicating the binding of the 3'-side phosphates with the P2 subsite of RNase A.

Subsite affinity of a glucoamylase from Aspergillus saitoi.

In order to investigate the subsite structure of the glucoamylase from Asp. saitoi, kinetic parameters (Km and ko values) of the enzyme for various sizes of maltooligosaccharide substrates were measured. The subsite affinities of this enzyme were calculated from the kinetic parameters and compared with those of the Rhizopus sp. glucoamylase, which differs from the Asp. saitoi glucoamylase in molecular weight, pI and of course, origin. The characteristic features of the subsite affinities of glucoamylase from Rhizopus, reported by Hiromi and his colleagues [Agric. Biol. Chem., 47, 573 (1983); Biochim. Biophys. Acta, 302, 362 (1973)], were retained in the glucoamylase from Asp. saitoi. However, the kint value for the glucoamylase from Asp. saitoi was about half of that for the enzyme from Rhizopus sp.

Dynamic evidence for an extended subsite structure of the ligand combining site on wheat germ agglutinin: temperature-jump relaxation with fluorescence detection

Temperature-jump relaxation methods have been used to study the binding kinetics of fluorescent 4-methylumbelliferyl glycosides of N-acetyl-beta-D-glucosamine and its beta (1 leads to 4)-linked di- and trisaccharides with wheat germ agglutinin. The mono- and disaccharide derivatives yielded biexponential progress curves. The data are consistent with two simple mechanisms in which binding occurs to an extended combining site on the lectin, consisting of at least two different, mutually exclusive, binding subsites. For one model, the bound ligand must slide from one subsite to the other, and the other mechanism requires the dissociation of the bound ligand from the protein before it can combine to the other subsite. Binding of 4-methylumbelliferyl monosaccharide to nonequivalent sites is improbable. The underlying kinetic and equilibrium parameters were obtained for the proposed subsites. The binding kinetics of the 4-methylumbelliferyl trisaccharide derivative are more complicated and may result from ligand-mediated linking reactions between molecules of the lectin. This study emphasizes that binding studies at equilibrium should take into account that the data result from an average of different binding configurations of all the ligands.

Subsite structure and ligand binding mechanism of glucoamylase.

1. The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. 2. The subsite structures of several amylases including glucoamylase were summarized. 3. In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration. 4. From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite 1 (nonreducing end side) where a tryptophan residue is located. 5. The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL complex. For substrates the EL may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.

タカアミラーゼＡと細菌糖化型α‐アミラーゼのサブサイト構造と転移作用

タカアミラーゼＡと細菌糖化型α‐アミラーゼのサブサイト構造と転移作用

Fractionation of isozymes and determination of the subsite structure of glucoamylase from Rhizopus niveus.

Four isozymes of glucoamylase were isolated from a commercial preparation of the enzyme from Rhizopus niveus by ion-exchange chromatography on CM-Sephadex C-50 and/or the preparative isoelectric focusing method on Sephadex IEF.The kinetic parameters, Michaelis constant Km and molecular activity k0, for the hydrolysis of maltooligosaccharides (degree of polymerization DP from 2 to 7) were determined on the major component enzyme at pFl 4.5 and 25°C. It was found that Km decreases and k0 increases with increasing DP. From the DP-dependences of Km and k0, the subsite affinities of the seven subsites of the enzyme active site were evaluated on the basis of subsite theory. These values are very similar to those obtained for an unfractionated preparation of Rhizopus delemar glucoamylase [Hiromi et al, B.B.A., 302, 362 (1973)].

α‐グルコシダーゼの多様性 基質特異性を中心に

The diversity of α-glucosidase was discussed from the viewpoint of substrate specificity. The substrate specificities of α-glucosidases from various origins were compared with one another on the hydrolysis velocity of α-glucosidic bond, calculated, from Vmax for the hydrolysis of substrate. It seems that many of α-glucosidases can be divided into the following three types of their substrate specificities, except an enzyme such as honey bee α-glucosidase having unusual properties. The first group (“Type I”) is the typical α-glucosidase which hydrolyzes heterogeneous substrates such as phenyl-α-glucoside and sucrose more rapidly than maltose. The second group (“Type II”) is the type of so-called maltase, showing especially high activity to homogeneous substrates such as maltooligosaccharides, but feeble or no activity to α-glucoside and sucrose. The third group (“Type III”) is characterized as α-glucosidases possessing glucoamylase activity. However, the substrate specificity of the third group may be classified into category of the second group, except that this type of a-glucosidases are capable of attacking α-glucans. The active site of α-glucosidase, like glucoamylase, was also shown to be made up by the subsite structure. The subsite affinities in the active site of buckwheat α-glucosidase, evaluated in accordance with the subsite theory, were compared with those of Rh, delemar glucoamylase. The difference in the substrate specificities between α-glucosidase and glucoamylase was interpreted on the basis of their subsite affinities.

Studies on the Subsite Structure of Amylases. III. Inhibition by Gluconolactone of the Hydrolysis of Maltodextrin Catalyzed by Glucoamylase1 from Rhizopus niveus2

Inhibition by gluconic acid-1 : 5-lactone (gluconolactone) and phenyl alpha-glucoside of the hydrolysis of maltodextrin catalyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus was investigated in relation to the subsite structure of the enzyme. Inhibition by gluconolactone was of the mixed type, whereas that by phenyl alpha-glucoside was purely competitive. These inhibition types were consistent with a theoretical prediction based on the assumption that gluconolactone and phenyl alpha-glucoside bind mainly to Subsites 1 and 2, respectively. The inhibitor constant of gluconolatone was determined to be 1.5 mM, which is in good agreement with the dissociation constant estimated by difference spectrophotometry (1.5 mM) (Ohnishi, M, et al. (1975) J. Biochem, 77, 695-703).

Studies on the subsite structure of amylases. IV. Tryptophan residues of glucoamylase from Rhizopus niveus studied by chemical modification with N-bromosuccinimide.

Chemical modification of glucoamylase [EC 3.2.1.3] from Rhizopus niveus by N-bromosuccinimide was carried out to investigate the role of tryptophan residues in the enzyme-catalyzed reaction and their location in the enzyme subsites. Of the ten tryptophan residues of the enzyme four could be modified. The two more reactive residues were confirmed not be essential for the catalytic activity for the hydrolysis of maltodextrin and phenyl alpha-maltoside. Complete loss of the catalytic activity, however, was brought about by modifying the two less reactive residues, and the modification of these residues was prevented by the substrates. The characteristic difference spectrum produced by maltose (7) disappeared in parallel with the loss of the catalytic activity. These results suggest that the tryptophan residue(s) responsible for the maltose-induced difference spectrum may be located at one of the subsite near the catalytic site and plays an important role in the catalytic activity of the enzyme.

Studies on the subsite structure of amylases. II. Difference-spectrophotometric studies on the interaction of maltotriose with liquefying alpha-amylase from Bacillus subtilis.

The difference spectra of liquefying alpha-amylase (EC 3.2.1.1) from B. subtilis upon the addition of a slowly reacting substrate, maltotriose, were measured to investigate specific binding of the substrate to the enzyme. The spectra produced by maltotriose were attributed to one tryptophan and one tyrosine residues on the basis of analysis of their shape and magnitude. From the dependence of the difference absorption upon the concentration of maltotriose, the dissociation constant of the maltotriose-enzyme complex was determined to be 170(+/- 20) mM, which is in good agreement with the Michaelis constant, Km obtained from the steady-state kinetics. The difference spectrum characteristic of a tryptophan residue was significantly decreased by the chemical modification of a trytophan residue with N-bromosuccinimide.

Studies on the subsite structure of amylases. I. Interaction of glucoamylase with substrate and analogues studied by difference-spectrophotometry.

Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1: 5-lactone (gluconolactone), methyl beta-D-glucoside, cellubiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or K1 obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the non-reducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue with shows a trough near 300 nm in difference spectra is located in this subsite.

A comparative study of NAD + binding sites in dehydrogenases by circular polarization of fluorescence

The spectra of the circular polarization of luminescence of a number of dehydrogenases with the fluorescent coenzyme nicotinamide-1,- N 6-ethenoadenine dinucleotide were measured. By use of this technique it is demonstrated that there is a difference in structure between the adenine subsite in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase on the one hand and pig heart lactate dehydrogenase, horse liver alcohol dehydrogenase, beef liver glutamate dehydrogenase, and pig heart malate dehydrogenase on the other hand. It is concluded that the non-co-operative dehydrogenases have similar, if not identical, adenine subsites whereas in glyceraldehyde-3-phosphate dehydrogenase, a strongly co-operative enzyme, a different structure of the adenine subsite has evolved.

Sequential Appearance of Three Different Anti-Fluorescein Combining Sites in Hyperimmunized Rabbits: Characterization by Circular Dichroism and Binding Studies

Abstract Fluorescein (F1) bound in the combining site of anti-F1 antibodies exhibits extrinsic optical activity which is a function of the fine structure of the site. We have utilized this phenomenon to study structural changes in antibody combining sites as a function of time and repeated boosting. Four rabbits were immunized with F1-keyhole limpet hemocyanin (KLH) and successively boosted whenever the antibody production declined. Antibodies were purified from bleedings taken once or twice a week and characterized by the extrinsic circular dichroism (CD) of their hapten-antibody complexes. After the third boost, three characteristically different CD spectra were observed, each corresponding to a different combining site configuration. Usually each individual bleed contained only one of the three antibody types and the particular type observed depended on the time after boosting: type I was present for the first 3 to 6 weeks of the antibody response, type II was present for a further 2 to 3 weeks and, finally, as the antibody production declined, only type III antibodies were found. This sequential response was repeated after each succeeding boost. Each of the antibody types was functionally homogeneous with respect to its extrinsic CD properties. In contrast, binding data showed that they were somewhat heterogeneous. The induced CD bands of bound F1 originate in the xanthenone ring whereas the binding of F1 depends on the interactions of both the xanthenone and phenylcarboxylate moieties with the site. Therefore, these results suggested that the anti-F1 combining site consists of two distinct subsites. The functional homogeneity of each antibody type may be due to a common tertiary structure of the xanthenone-binding subsite whereas the heterogeneity indicated by the binding data may be due to heterogeneity within the phenylcarboxylate-binding subsite. Possible mechanisms for the sequential appearance of these antibody types are also discussed.

アミラーゼの作用様式とサブサイト構造

アミラーゼの作用様式とサブサイト構造