The role of carboxyl, guanidine and imidazole groups in catalysis by a midgut trehalase purified from an insect larvae

• Published in: Insect biochemistry and molecular biology 2004-10, Vol.34 (10), p.1089-1099
• Main Authors: Silva, Maria C.P., Terra, Walter R., Ferreira, Clélia
• Format: Article
• Language: English
• Subjects: active sites; Animals; binding sites; Binding subsites; Binding, Competitive; catalysts; Catalytic arginine residue; Catalytic Domain; Chemical modification; Digestive System - enzymology; enzyme activity; enzyme inhibitors; Enzyme Inhibitors - pharmacology; enzyme kinetics; Hydrogen-Ion Concentration; Insect control; Kinetics; Larva - enzymology; larvae; Lepidoptera; midgut; Midgut trehalase; Molecular Structure; Noctuidae; purification; Solubility; Spodoptera - enzymology; Spodoptera frugiperda; trehalase; Trehalase - antagonists & inhibitors; Trehalase - chemistry; Trehalase - isolation & purification; Trehalase - metabolism; Trehalose
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2004.07.001

A trehalase (EC 3.2.1.28) of 67 kDa was purified to homogeneity from the midgut of Spodoptera frugiperda (Lepidoptera) larvae. The enzyme is inhibited by toxic β-glucosides produced by plants (amygdalin, prunasin, salicin and phlorezin) and by their aglycones (mandelonitrile, phloretin). From k cat and K m values determined in different pHs, the p K a values of catalytic essential groups were calculated (p K a=4.5 and p K a=8.0). These p K a values agree with the ones determined from enzyme chemical in activation with carbodiimide and phenyl glyoxal, respectively, indicating that the enzyme has a carboxyl group that act as a nucleophile and a guanidine group that is the proton donor during the catalytic cycle. The enzyme has two putative subsites for glucose binding. Based on the protection afforded by ligands against chemical modification, the roles of the subsites were inferred. Thus, the one that binds the competitive inhibitors, methyl α-glucoside (MαGlu) and mandelonitrile, contains the catalytic carboxyl, whereas the other having the catalytic Arg residue binds the competitive inhibitor Tris. Diethyl pyrocarbonate is ineffective except in the presence of MαGlu, when it decreases trehalase activity and changes the p K a value of the catalytic Arg residue. This suggests that the p K a value of the Arg residue is modulated by a His residue located near the active site. This also indicates that the enzyme molecule changes its conformation when the subsite containing the carboxyl group is occupied. The increase in trehalase inactivation by phenyl glyoxal in the presence of MαGlu agrees with the last observation.