Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions.
The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)
Omichi
K
K
Department of Chemistry, Osaka University College of Science.
Shiosaki
K
K
Matsubara
K
K
Ikenaka
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004586:Electrophoresis; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012463:Saliva / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014961:X-Ray Diffraction; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D000516:alpha-Amylases