Partial amino acid sequences around sulfhydryl groups of soybean beta-amylase

Sulfhydryl (SH) groups of soybean beta-amylase were modified with 5-(iodoaceto-amidoethyl)aminonaphthalene-1-sulfonate (IAEDANS) and the SH-containing peptides exhibiting fluorescence were purified after chymotryptic digestion of the modified enzyme. The sequence analysis of the peptides derived from the modification of all SH groups in the denatured enzyme revealed the existence of six SH groups, in contrast to five reported previously. One of them was found to have extremely low reactivity toward SH-reagents without reduction. In the native state, IAEDANS reacted with 2 mol of SH groups per mol of the enzyme (SH1 and SH2) accompanied with inactivation of the enzyme owing to the modification of SH2 located near the active site of this enzyme. The selective modification of SH2 with IAEDANS was attained after the blocking of SH1 with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid sequences of the peptides containing SH1 and SH2 were determined to be Cys-Ala-Asn-Pro-Gln and His-Gln-Cys-Gly-Gly-Asn-Val-Gly-Asp-Ile-Val-Asn-Ile-Pro-Ile-Pro-Gln-Trp, respectively.     

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.     

The effect of modification of sulfhydryl groups in soybean lipoxygenase-1

Soybean lipoxygenase-1 was found to contain five free sulfhydryl groups and no disulfide bridges. Three sulfhydryl groups react readily with methylmercuric halides. This modification results in significant changes of the catalytic properties of the enzyme. Comparison of modified and native lipoxygenase-1 shows the following: 1. The catalytic constant of the oxygenation of linoleic acid is reduced by approximately 50%, whereas the affinity towards linoleic acid remains unaltered. 2. At high concentrations of substrate and low concentrations of enzyme the kinetic lag phase in the oxygenation is considerably longer. 3. The regio- and stereospecificities of the oxygenation are significantly lower. 4. Besides hydroperoxides, oxo-octadecadienoic acids (4%) are formed during the oxygenation. 5. The cooxidation capacity is considerably enhanced. Treatment of methylmercury-modified lipoxygenase-1 with NaHS results in the complete recovery of the sulfhydryl groups and of the catalytic properties.     

Vapor-phase modification of sulfhydryl groups in proteins

Proteins and peptides are readily and specifically modified at their sulfhydryl groups by the vapors of a mixture of 4-vinylpyridine and tributylphosphine. The phenylthiohydantoin derivative of S-beta-(4-pyridylethyl)cysteine formed during sequence analysis is easily detectable in current identification systems.     

Chemical modification of Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups

Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.     

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups.

Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).     

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.     

N-terminal sequence of soybean beta-amylase

The blocked N-terminus and N-terminal sequence of soybean beta-amylase were determined by analyzing the acidic peptides derived on peptic digestion of the enzyme. The acidic peptides were separated from the digest on a Dowex 50 X 2 column and purified by reversed phase-high performance liquid chromatography (RP-HPLC). The major acidic peptide, Pep-4, was a heptapeptide with a molecular weight of 766. Forty-eight hundredths mol acetyl group and 0.61 mol acetyl-Ala per mol of Pep-4 were detected on RP-HPLC analysis. The N-terminal 9 amino acid sequence of soybean beta-amylase was deduced to be acetyl-Ala-Thr-Ser-Asp-Ser-Asn-Met-(Gly-Leu) from the results of sequence analysis of Pep-4 and amino acid analysis of other acidic peptides.     

Chemical modification of sulfhydryl residues of rabbit triosephosphate isomerase

Rabbit muscle triosephosphate isomerase (EC 5.3.1.1) is inactivated by maleimides, Na₂S₄O₆, organic mercurials, 5,5′-dithiobis (2-nitrobenzoic acid), Ag⁺, and Hg²⁺. Ag²⁺ and Hg²⁺ cause a decrease in the maximum velocity, and under specified conditions the other reagents induce an increase in the Michaelis constant.N-ethylmaleimide reacts with three sulfhydryl residues per mole of enzyme, and the maximum change in K_m is about threefold. Mercurials cause a greater change in K_m and react with more than three sulfhydryl groups, but subsequent precipitation prevents quantitative analysis after six residues have reacted (with p-hydroxymercuribenzoate).Experiments with several competitive inhibitors and the active-site affinity label, 3-chloroacetolphosphate, showed that the magnitude of the change in Michaelis constant was the same as the magnitude of the changes in the inhibition constants.The rabbit muscle and liver enzyme appear to have similar properties, but the chicken muscle enzyme is much less reactive, and the yeast enzyme does not become inactivated.Evidence is presented to show that the effects cannot be explained by assuming the hydrated substrates are bound to the enzyme as a result of sulfhydryl modification.     

Interaction of soybean beta-amylase with glucose

The interaction of soybean beta-amylase with glucose was investigated by inhibition kinetics studies and spectroscopic measurements. The inhibition type, inhibitor constant (Ki) and dissociation constant (Kd) of beta-amylase-glucose complex were dependent on pH. At pH 8.0, glucose behaved as a competitive inhibitor (Ki = 34 mM). Binding of glucose produced a characteristic difference spectrum and a change of circular dichroism (CD) at pH 8.1. By using difference absorbance at 292 nm and difference ellipticity at 290 nm, Kd values for beta-amylase-glucose complex were determined to be 45 and 46 mM, respectively. In contrast to pH 8.0, glucose behaved as a mixed-type inhibitor (Ki = 320 mM) at pH 5.4. The Kd values obtained from the difference spectrum were increased by lowering the pH from 8. The pH dependence of the Ki and Kd values suggested that one ionizable group of pK = 8.0, which is shifted to 6.9 by the binding of glucose, controls the binding affinity of glucose. The binding of glucose competed with the binding of cyclohexaamylose and maltose at pH 8.0. The modification of SH groups of the enzyme affected the binding of glucose but did not affect the binding of maltose or cyclohexaamylose at pH 8.0. It was concluded from these results that the binding site of glucose is different from that of maltose and cyclohexaamylose. Presumably, glucose may bind to the subsite 1 of soybean beta-amylase.     

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.     

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.     

Chemical modification of corn fiber with ion-exchanging groups

Corn fiber was chemically modified with ion-exchanging groups to prepare water-soluble polysaccharides. The soluble fractions were dialyzed using dialysis tubing (1 kDa) and the material retained inside the tubing was filtered through 10 kDa membranes to separate into fractions with molar mass of 1–10 kDa and greater than 10 kDa. The yield of solubilized material of molar mass higher than 10 kDa (47%) and 1–10 kDa (17%) obtained by sulfonation in the presence of NaOH under vacuum was greater than the yields of the treatment at the ambient pressure (43% and 2%) and also in experiments run with only KOH (40% and 5%) or NaOH (38% and 5%) at ambient pressure. The sugar analysis indicated that they were typical glucuronogalactoarabinoxylans containing 46–57% d-xylose (Xyl), 25–33% l-arabinose (Ara) and 6–12% d-galactose (Gal).