The role of carboxyl, guanidine and imidazole groups in catalysis by a midgut trehalase purified from an insect larvae.
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 beta-glucosides produced by plants (amygdalin, prunasin, salicin and phlorezin) and by their aglycones (mandelonitrile, phloretin). From kcat and Km values determined in different pHs, the pKa values of catalytic essential groups were calculated (pKa = 4.5 and pKa = 8.0). These pKa 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 alpha-glucoside (MalphaGlu) 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 MalphaGlu, when it decreases trehalase activity and changes the pKa value of the catalytic Arg residue. This suggests that the pKa 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 MalphaGlu agrees with the last observation.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-200 São Paulo, Brazil.
Maria C P
MC
Silva
Walter R
WR
Terra
Clélia
C
Ferreira
D000818:Animals; D001667:Binding, Competitive; D020134:Catalytic Domain; D004064:Digestive System / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D015394:Molecular Structure; D012995:Solubility; D018411:Spodoptera / Q000201:enzymology*; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014198:Trehalase