Residues essential for catalytic activity of soybean β‐amylase

Abstract

To determine which amino acid residues are essential for the catalytic activity of soybean P-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in P-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue.Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean P-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in /I-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30"C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.8-Amylase (1 ,4-a-D-glucan maltohydrolase) hydrolyzes a-l,4-glucosidic linkage of starch and glycogen with liberation of P-anomeric maltose from the non-reducing ends. This enzyme is found in many plant and bacteria species. Although the physicochemical properties of the enzymes from sweet potato, barley, and soybean have been well investigated [l], the catalytic mechanism of the enzyme has not yet been elucidated. Comparison of deduced sequences of P-amylases originating from plant and bacteria revealed three highly conserved sequences which very likely are involved in the segment assembly of the catalytic site of /I-amylase [2-41. In the case of a-amylase (1,4-a-~-glucan glucanohydrolase) that catalyzes the hydrolysis of 1,4-a-D-glucans, the aspartyl and glutamyl residues contained in highly conserved regions have been identified as catalytic residues by X-ray crystallography [5 -71 and by site-directed mutagenesis [8, 91. Although the action pattern of P-amylase differs from that of a-amylase and there is no significant similarity between the primary sequences, all of the aspartyl and glutamyl residues located in the conserved regions of P-amylase family can be considered possible catalytic residues. Recently, the results of affinity labeling of soybean P-amylase with 2,3-epoxy propyl a-D-glucopyranoside suggested that Glu186 was involved in the catalytic site [lo]. In addition, since the enzyme was inactivated by sulfhydryl reagents such as iodoacetamide, N-ethylmaleimide and mercuribenzoate [ l , 111, the cysteinyl residues located in the regions may also participate in the catalytic reaction.

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

“…Failure to remove this intron would cause a frameshift in the middle of the coding region. Site-directed mutagenesis experiments indicate that at least one amino acid within the region that would be affected by this frameshift plays a critical role in ␤-amylase activity (Totsuka et al, 1994). In addition, x-ray crystallographic data reveal that amino acids that would be affected by this frameshift form part of the region of the protein comprising the catalytic center (Mikami et al, 1994).…”

“…The Y residue is particularly well conserved, because it is present not only in ␤-amylases from plants but also in several species of bacteria (Totsuka et al, 1994). Mutating the C residue present at the equivalent position in a ␤-amylase from soybean did not result in a significant reduction in ␤-amylase activity (Totsuka et al, 1994). However, because the four amino acids predicted to be missing from the ␤-amylase produced by ram1 are highly conserved and include two that are charged, the removal of all four amino acids is likely to result in severe alterations in protein structure and activity.…”

“…Although these four amino acids are not believed to interact directly with the ␤-amylase substrate (Mikami et al, 1994), a search of the literature (Totsuka et al, 1994) and of the GenBank database reveals that these amino acid residues are highly conserved among ␤-amylases from a variety of plant species. The Y residue is particularly well conserved, because it is present not only in ␤-amylases from plants but also in several species of bacteria (Totsuka et al, 1994). Mutating the C residue present at the equivalent position in a ␤-amylase from soybean did not result in a significant reduction in ␤-amylase activity (Totsuka et al, 1994).…”

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

“…Chemical Rescue of the Inactive E294A Mutant-Although the substantial decrease in the catalytic activity of the mutant enzyme lacking the putative nucleophilic residue is consistent with its suggested role, the identification of the catalytic residues could not be based on the reduction in activity alone. There are several examples where single mutations in proposed catalytic residues of glycoside hydrolases resulted in reduced or undetectable activity (46,47), whereas crystallographic and biochemical analyses showed that these residues are not involved directly in catalysis (48,49). To unambiguously identify Glu-294 as the nucleophilic residue of AbfA T-6, the azide rescue methodology was used.…”

Functional Analysis of Glu380 and Leu383 of Soybean β‐Amylase

“…Since [Leu383Tle] p-amylase and [Leu383Gln]p-amylases were not adsorbed onto a cyclomaltohexaose-immobilized Sepharose 6B column, the enzymes were further fractionated on a Mono S HR 10110 column (Pharmacia Biotech AB) with a linear gradient of sodium chloride of 0-0.5 M in 50 mM sodium acetate, pH 5.0, i n the final stcp of purification. In the case of inactive mutants, each purification step was monitored by immunoblotting [14].…”