Koichi Honda

Novel substrate specificity of a membrane-bound β-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A β‐glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X‐100 at 85°C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100°C, respectively. BGPh stability was dependent on the presence of Triton X‐100, the enzymes half‐life at 90°C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k cat/K m values high enough for hydrolysis of β‐D‐Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of β‐linked glucose dimer more hydrophilic than aryl‐ or alkyl‐β‐D‐Glcp.

An increase in the transglycosylation activity of Saccharomycopsis α-amylase altered by site-directed mutagenesis

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

A mutant α-amylase with enhanced activity specific for short substrates

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera α‐amylase was altered to arginine (R) or asparagine (N) by site‐directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three‐times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.

Multi-functional roles of a histidine residue in human pancreatic α -amylase

Abstract Functional roles of histidine residues at the active site in human pancreatic α -amylase were examined by protein engineering. Three histidine residues at 101, 201, and 299 were converted to asparagine residues, respectively. It was found that His201 played multi-functional roles concerning so many functions; substrate binding, control of optimum pH, change in substrate specificity, activation by chloride ion, and inhibition by a proteinaceous inhibitor.

The pH dependence of the action pattern in porcine pancreatic α-amylase-catalyzed reaction for maltooligosaccharide substrates

Porcine pancreatic alpha-amylase (EC 3.2.1.1; abbreviated PPA), which hydrolyzes alpha-D-(1,4) glucosidic bonds in starch and amylose, displays an optimum at pH 6.9 for the majority of substrates. The optimum pH, however, shifted to 5.2 for the hydrolysis of some low molecular substrates (Ishikawa, K., et al., 1990, Biochemistry 29, 7119-7123). Details of the substrate-dependent shift of the optimum pH in PPA were studied by use of a series of maltooligosaccharides with 14C-labeled reducing end glucose as substrates. The optimum pH for maltotriose was 5.2, whereas that for maltopentaose and maltohexaose was unchanged at pH 6.9. The pH profile for the intermediate size substrate maltotetraose showed abnormality; the apparent optimum pH was broadened between 5.5 and 6.5 and the bond cleavage pattern depended on pH, unlike that for the other substrates examined. These results were independent of either buffer systems or substrate concentration. Analyses of the hydrolysates of the maltooligosaccharides revealed that the shift of the optimum pH to the neutral region occurred only when the fifth subsite of PPA in the productive binding modes was occupied by a glucosyl residue of a substrate. The three-catalytic residue model of PPA deduced from the analysis of the hydrolysis of some modified maltooligosaccharides (p-nitrophenyl-alpha-D-maltoside, gamma-cyclodextrin, maltopentaitol, and maltohexaitol) (Ishikawa, K., et al., 1990, Biochemistry 29, 7119-7123) was successfully adapted to the linear maltooligosaccharides used in this work. These results indicate that the different productive binding modes of the linear oligosaccharide substrates affect directly the catalytic power and the optimum pH of PPA.

Conversion of alpha -amylase into transglycosylation enzyme

The 210 th lysine residue in Saccharomycopsis α-amylase (Sfamy) molecule was replaced by arginine and asparagine residues. The resulting K210R and K210N enzymes cleave mainly the first glycosidic bond from the reducinng end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G4. We estimated the 8th subsite affinities of the mutant enzymes and compared them with that of the native enzyme. These facts suggest that the K210 residue composes the 8th subsite, one of the major subsites, and that a positively charged s-amino residue is necessary for the 8th subsite affinity. The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite. The 84th tryptophan residue was replaced by leucine residues. The resulting W84L enzyme showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. These large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the W84 residue located at subsite 5 plays an important role in the addition of a water molecule to a carbonium ion intermediate and/or in the liberation of the hydrolysis product from the substrate binding pocket. The doubly mutated enzymes, W84LK210 N, are expected to form the transglycosylation products different in size from those produced by the single mutant, W84L enzyme.