Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.
High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.
Frandsen
T P
TP
Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Lok
F
F
Mirgorodskaya
E
E
Roepstorff
P
P
Svensson
B
B
D000595:Amino Acid Sequence; D018525:Germination; D005960:Glucosides / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology* / Q000254:growth & development; D006868:Hydrolysis; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D013237:Stereoisomerism; D000520:alpha-Glucosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D005960:Glucosides; D000520:alpha-Glucosidases