The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda.
Trehalase (EC 3.2.1.28) hydrolyzes only α, α'- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has D312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (D322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity.
Silva
Maria C P
MC
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
Terra
Walter R
WR
Ferreira
Clélia
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D004064:Digestive System / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018411:Spodoptera / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014198:Trehalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014199:Trehalose / Q000378:metabolism
D019476:Insect Proteins; D014199:Trehalose; D014198:Trehalase