Difference spectroscopic study of the interaction between soybean beta-amylase and substrate or substrate analogues

1. In order to investigate the interactions between soybean beta-amylase [EC 3.2.1.2] and ligands (maltotriose as substrate, and maltose and alpha- and beta-cyclodextrins as inhibitors for the hydrolysis of maltoheptaose), the difference spectra were measured at 25 degrees C and pH 5.4, in 0.05 M acetate buffer. Each difference spectrum produced by these ligands showed a clear peak at 292-293 nm due to a tryptophan residue. In addition to this peak, the spectra of alpha- and beta-cyclodextrins showed a specific peak at 298-299 nm, and that of maltotriose showed a shoulder at 298 nm. 2. From the concentration dependency of the difference molar extinction delta epsilon, at 292-293 nm or at 298-299 nm, the dissociation constant of the enzyme-ligand complex, Kd, was evaluated for maltotriose, and alpha- and beta-cyclodextrins. For each ligand, the Kd values obtained at these two wavelengths were in good agreement with Michaelis constant, Km, or the inhibitor constant, Ki. The Kd value for maltose obtained from the titration of delta epsilon at 292 nm was also in good agreement with Ki. 3. Maltose produced a hydrophobic change in the environment of the tryptophan residue, while the interactions of maltotriose, and alpha- and beta-cyclodextrins with this enzyme caused an electrostatic change in the vicinity of the tryptophan residue in addition to the hydrophobic change. Since the signal at 298-299 nm was not found in the difference spectrum of maltose, this signal may be due to a tryptophan residue different from that which produces the signal at 292-293 nm. If both the signals are due to the same tryptophan residue, we must conclude that some conformational change is caused in the enzyme active site by the ligand binding.     

Effect of modification of sulfhydryl groups in soybean beta-amylase on the interaction with substrate and inhibitors

Methyl 2,4-dinitrophenyl disulfide (MDPS) is shown to be an effective methanethiolating reagent for sulfhydryl groups in proteins via thiol-disulfide exchange reaction. It reacts with the two reactive sulfhydryl groups (SH1 and SH2) in soybean beta-amylase. A decrease of the enzymatic activity accompanies the methanethiolation of SH2. After complete methanethiolation of SH2, the modified enzyme still has 9% of the initial activity. Modification of SH2 with cyanide and iodoacetamide reduces the enzymatic activity to 65 and 2% of the initial activity, respectively. Apparently, the residual activity depends upon the size of the substituent at SH2. The modified enzymes still have the almost same Km values for amylopectin and Kd values for enzyme-maltose and enzyme-cyclohexaamylose complexes as the native enzyme. In contrast to maltose and cyclohexaamylose, the Kd value of the enzyme-glucose complex increases in the order of cyanide-, MDPS-, and iodoacetamide-modified enzymes, indicating that SH2 is located near the binding site of glucose. It is proposed from the subsite structure of soybean beta-amylase that the position of SH2 and the glucose binding site is around subsite 1, where the nonreducing ends of the substrate bind productively.     

Hydrolysis of aryl beta-maltotriosides by sweet potato beta-amylase and soybean beta-amylase.

Sweet potato beta-amylase [EC 3.2.1.2, alpha 1,4-D-glucan maltohydrolase]-catalyzed hydrolyses of aryl beta-maltotriosides with substituents, NO2-, Cl-, and Br- at the o-, m-, and p-positions in the phenyl ring were studied at pH 4.8 and 25 degrees C. The hydrolyses of a few of the maltotriosides by soybean beta-amylase [EC 3.2.1.2, alpha-1,4-D-glucan maltohydrolase] were also studied at pH 5.4 and 25 degrees C. It was found that the aryl beta-maltotriosides were preferentially hydrolyzed into maltose and aryl beta-D-glucosides by both beta-amylases. The Michaelis constant Km and the molecular activity ko were determined for the hydrolyses of these maltotriosides and compared with those of maltotriose and maltotetraose. Aryl beta-maltotriosides were more rapidly hydrolyzed than maltotriose by a factor of 30--80, and more slowly hydrolyzed than maltotetraose by a factor of 10--30, depending on the kinds of substituents. The rapid hydrolysis of aryl beta-maltotrioside as compared with maltotriose may be due to the interaction of an aryl group with the subsite of beta-amylase. This is in contrast with glucoamylase [EC 3.2.1.3, alpha-1,4-D-glucan glucohydrolase] of Rhizopus niveus-catalyzed hydrolysis of phenyl beta-maltoside, whose phenyl group does not interact so much with the subsite of the enzyme.     

Crystallization and preliminary x-ray investigation of soybean beta-amylase.

Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52.     

Reversible immobilization of soybean beta-amylase on phenylboronate-agarose

Soybean beta-amylase (EC 3.2.1.2) wap immobilized on phenylboronate-agarose by strong interactive binding. The insoluble derivative was active and more stable to temperature changes than the free enzyme. The absence of enzyme leakage even in the presence of substrate was demonstrated. Changes in pH over a wide range (4.0-8.0) did not affect the stability of the complex. The support could be recovered by sorbitol elution, which demonstrated the reversibility of the binding. Since the enzyme was not retained on phenylagarose under similar conditions, we rejected hydrophobic interactions as a cause of the strong binding of the enzyme to phenylboronate-agarose. We suggest that the bonding of the enzyme to the phenylboronate ligand occurs by a charge transfer mechanism between the trigonal boronate and the side chain nitrogenated groups. It was concluded that phenylboronate-agarose has good properties as a support, which recommends its use for the preparation of immobilized enzymes.     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Anatomy of a conformational transition of beta-strand 6 in soybean beta-amylase caused by substrate (or inhibitor) binding to the catalytical site.

A computational study of the five soybean beta-amylase X-ray structure reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the beta-strand 6 (residues 341-343) in the (beta/alpha)8 molecular scaffold. Backbone distortions that involve considerable changes in the phi and psi angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA-layer (at the C-terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand-binding process, there is a negative enthalpy change for these residues (-27 and -34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed "stability-function" hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452-456). They are also in good agreement with other experimental results in the literature that describe the role of the 341-343 segment in beta-amylase activity. Site-directed mutagenesis focused on these residues could be useful for undertaking functional studies of beta-amylase.     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

Studies on the interaction between actin and cofilin purified by a new method.

Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.     

荧光法研究茜素红S与壳聚糖作用机理及其应用

应用荧光法研究了茜素红S和壳聚糖之间相互作用的机理,测得Kq=2.9581×1011L.(mol.s)-1;KA=3.26×103;n=102。建立了测定壳聚糖含量的茜素红S荧光猝灭方法,线性回归方程为F0/F=-0.43837+0.43627C(mg.L-1),线性范围为0.000～16.667mg.L-1,检测限为0.473;平均回收率为100.60%。     

Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site

Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center.     

Location of SH groups along the polypeptide chain of soybean beta-amylase

The five SH groups of soybean beta-amylase differ in reactivity toward SH reagents such as 2,2'-dithiopyridine (2-PDS), monoiodoacetate and N-ethylmaleimide (NEM). They were designated as SH1, SH2, SH3, SH4, and SH5, in order of their reactivity except for the two buried SH groups, SH4 and SH5. The location of the five SH groups along the polypeptide chain was determined by specific cleavage at the amino side of their cyanocysteine residues which were formed by converting SH to SCN groups by cyanide after modifying the SH groups with 2-PDS. The selective modification of SH groups was achieved as follows: SH1 reacted with 2-PDS at low and high ionic strength, while SH2 reacted only at high ionic strength. SH2 and SH3 were also modified with 2-PDS using SH1-carboxymethylated soybean beta-amylase. The buried SH groups, SH4 and SH5, were modified with 2-PDS under the denaturation conditions after the reactive SH groups, SH1, SH2, SH3, were irreversibly blocked with NEM. On the other hand, the five SH groups were cyanylated with [14C]cyanide or with 2-nitro-5-thiocyanobenzoic acid (NTCB) for the cleavage at all five SH groups. The molecular weight estimation of derivatives of cleaved soybean beta-amylase by SDS-gel electrophoresis showed that the five pairs of fragments (Mw 50,000 & 6,500, 47,000 & 8,000, 38,000 & 18,000, 35,000 & 23,000, and 31,000 & 25,000) were identified with the fragments formed by cleavage at SH1, SH2, SH3, SH4, and SH5, respectively. By considering fragments incorporating 14C (Mw 47,000, 35,000, 25,000, 18,000, and 6,500), the fragments were aligned along the polypeptide chain of soybean beta-amylase, in order from the N-terminus as SH2, SH5, SH3, SH4, and SH1. This order is supported by estimating the molecular weight of fragments formed by high-yield cleavage using NTCB and by analyzing the COOH-terminal residues of the fragment cleaved at SH2.     

Identification of glutamic acid 186 affinity-labeled by 2,3-epoxypropyl alpha-D-glucopyranoside in soybean beta-amylase

Soybean beta-amylase was modified with 2,3-epoxypropyl alpha-D-[U-14C]glucopyranoside ([14C]alpha-EPG), a radioactive affinity-labeling reagent for beta-amylase, until it lost 95% of its enzyme activity. After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC. A radioactive peptide was finally isolated and its amino acid sequence was determined to be 181Leu-Gly-Pro-Ala-Gly-Glu186. Radioactivity derived from [14C]-alpha-EPG was found exclusively at Glu-186, the gamma-carboxyl group of which is esterified with the affinity label. It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean beta-amylase.     

Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.