Kenji Takase

Site-directed mutagenesis of active site residues in Bacillus subtilis α-amylase

Site-directed mutagenesis of Bacillus subtilis N7 α-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an active catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15 000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of k cat with a 5-fold increase in K m for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaoe were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic α-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutant by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could not be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.

A sedimentation equilibrium study of the temperature-dependent association of bovine β-casein

The temperature-dependent association of beta-casein was studied by the sedimentation equilibrium method. The weight-average molecular weight of the protein was determined in 0.2 M sodium phosphate buffer (pH 6.7) at 20, 15, 10 and 2 degrees C, and was found to be dependent markedly on both temperature and protein concentration. The data were well fitted by a monomer-n-mer association scheme, and the values of n, the second viral coefficient, and the free energy change for the association (association constant) were evaluated. The results show that temperature not only shifts the equilibrium but alters the polymer size: the values of n are 49, 22, and 12 at 20, 15, and 10 degrees C, respectively; the free energy change per monomer becomes more negative with increasing temperature, indicating that the attraction between the monomers in a polymer is stronger at higher temperature. This effect of temperature is in contrast to that of ionic strength which affects mainly the equilibrium. The enthalpy change, entropy change, and heat capacity change for the association were also estimated and were indicative of the formation of a considerable amount of hydrophobic bonds upon association.

Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose

The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.

Interaction of catalytic-site mutants of Bacillus subtilis α-amylase with substrate and acarbose

The interactions of the three catalytic-site mutants of Bacillus subtilis alpha-amylase/(DN176 [Asp-176----Asn], EQ208 [Glu-208----Gln] and DN269 [Asp-269----Asn]) with substrates and a pseudo-oligosaccharide inhibitor, acarbose, have been studied by means of difference absorption spectroscopy and affinity chromatography. The addition of maltopentaose or soluble starch to the inactive mutant enzymes mostly resulted in difference spectra characteristic of tryptophan perturbation, enabling determination of the dissociation constants. The results show that conversion of Glu-208 to Gln greatly enhanced substrate binding, implying that Glu-208 interacts unfavorably with the substrates ground state, preventing its optimal fit to the active site. The affinity for acarbose was greatly reduced in DN269 and EQ208, but less so in DN176, suggesting that Asp-269 and Glu-208 are more important than Asp-176 in stabilizing the transition state. These results are consistent with Glu-208 and Asp-269 being the key catalytic residues, as proposed for Taka-amylase A.

Artificial insertion of peptides between signal peptide and mature protein : effect on secretion and processing of hybrid thermostable α-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.

Thermodynamics of the unfolding of α-lactalbumin by guanidine thiocyanate

Abstract The reversible unfolding of α-lactalbumin by guanidine thiocyanate has been studied at 25.0 °C by means of difference spectra and optical rotation measurements. At pH 7.35, two tryptophanyl residues buried in the interior of the protein in the native state were considered to be partly exposed on its surface in the unfolded state. The dependence of the equilibrium constant of the unfolding reaction in aqueous solutions of guanidine thiocyanate on pH can be described in terms of the abnormal histidyl residues and indicated the protein is in the most stable state at pH 7.30. The dependence on the denaturant activity, which was obtained by the isopiestic method, results in a similar difference between the numbers of the binding sites on the unfolded and the native protein molecules to that in the case of the unfolding by guanidine hydrochloride, a larger intrinsic binding constant of the denaturant with the protein than that of the guanidine hydrochloride, and a more unstable structure in the native state than that of lysozyme. Also the dependence on the denaturant concentration results in the higher degree of exposure of both peptide units and hydrophobic side chains in the native state to solvent than in lysozyme.

Purification, crystallization and preliminary X-ray crystallographic study of α-amylase from Bacillus stearothermophilus

A recombinant alpha-amylase from Bacillus stearothermophilus was found to be produced as several isoforms arising from different N--terminal processing. Some of those isoforms were purified to homogeneity and crystallized at 293 K using the hanging-drop vapour-diffusion method under the following conditions: 35 mM sodium acetate (pH 4.6), 6.25%(v/v) 2-propanol, in the presence of 1.23%(w/v) acarbose (a pseudo-oligosaccharide inhibitor) in the drop. The crystals diffracted beyond 2.0 A resolution using synchrotron radiation at the Photon Factory, Tsukuba. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 53.7 (2), b = 92.9 (4), c = 53.2 (2) A, beta = 109.4 (1) degrees.