The role of residues R97 and Y331 in modulating the pH optimum of an insect β‐glycosidase of family 1

Abstract

The activity of the digestive b-glycosidase from Spodoptera frugiperda (Sfbgly50, pH optimum 6.2) depends on E399 (pK a ¼ 4.9; catalytic nucleophile) and E187 (pK a ¼ 7.5; catalytic proton donor). Homology modelling of the Sfbgly50 active site confirms that R97 and Y331 form hydrogen bonds with E399. Site-directed mutagenesis showed that the substitution of R97 by methionine or lysine increased the E399 pK a by 0.6 or 0.8 units, respectively, shifting the pH optima of these mutants to 6.5. The substitution of Y331 by phenylalanine increased the pK a of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed DpK a it was calculated that R97 and Y331 contribute 3.4 and 4.0 kJAEmol )1 , respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187 by D decreased the pK a of residue 187 by 0.5 units and shifted the pH optimum to 5.8, suggesting that an electrostatic repulsion between the deprotonated E399 and E187 may increase the pK a of E187, which then becomes the catalytic proton donor. In short the data showed that a network of noncovalent interactions among R97, Y331, E399 and E187 controls the Sfbgly50 pH optimum. As those residues are conserved among the family 1 b-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 b-glycosidases.Keywords: b-glycosidase; pK a values; pH optimum; sitedirected mutagenesis; Spodoptera frugiperda.The b-glycosidases from glycoside hydrolase family 1 are enzymes that remove monosaccharides from the nonreducing end of di-and/or oligosaccharides. According to the CAZy website this family comprises 422 sequenced b-glycosidases, of which the tertiary structure of 12 has been determined. Together with families 2, 10,17,26, 30, 35, 39, 42, 51, 53, 59, 72, 79 and 86 family 1 forms clan A, a group of families that shares structural and catalytic similarities [1]. All b-glycosidases of family 1 present the same tertiary structure [the (b/a) 8 barrel], they are configuration-retaining glycosidases and their catalytic activity depends on two glutamic acid residues, one positioned after b strand 4 and the other after b strand 7 [1]. These glutamic acids are very close inside the active site (about 4.5 Å apart) [2], and during the reaction the first glutamic acid acts as proton donor, and the second acts as a nucleophile. The catalytic nucleophile pK a is around 5.0 and the catalytic proton donor pK a is around 7.0 [3][4][5][6][7].A plot of b-glycosidase activity vs. pH presents a bell shape, indicating that in the pH optimum the catalytic nucleophile is deprotonated and the catalytic proton donor is protonated. Hence the branch of the curve below the pH optimum is determined mainly by the ionization of the catalytic nucleophile, whereas the catalytic ionization of the proton donor determines the branch above the pH optimum.As the b-glycosidase activity depends on the finely tuned ionization of the catalytic nucleophile and proton ...

Purification, characterization and sequencing of the major β-1,3-glucanase from the midgut of Tenebrio molitor larvae

“…Nevertheless, the enzyme has only one Arg residue in its active site, since the substitution of Arg 97 by Met or Lys leads to an enzyme that is not affected by PG. It is possible that in the conditions employed in this paper and in experiments done by Marana et al (2003), the binding of only one PG molecule is enough to inactivate the enzyme. If the complex of enzyme with one PG molecule is less stable than the complex with two PG molecules, the reaction order may be a number between 1 and 2, provided that on dissociating the enzyme-PG complex results in an active enzyme.…”

“…This work quantified incorporated PG, and did not determine the order of reaction. Marana et al (2003) modified a b-glycosidase with PG and obtained a reaction order of 1.7. Nevertheless, the enzyme has only one Arg residue in its active site, since the substitution of Arg 97 by Met or Lys leads to an enzyme that is not affected by PG.…”

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

“…It has been described that, depending on the conditions of the reaction, one or two PG molecules react with one Arg residue (see Mizohata et al, 2003). Marana et al (2003) modified a b-glycosidase with PG and obtained a reaction order of 1.7. Nevertheless, the enzyme has only one Arg residue in its active site, since the substitution of Arg97 by Met or Lys leads to an enzyme that is not affected by PG.…”

Caracterização funcional dos resíduos centrais da rede estrutural da β-glicosidase Sfβgli de Spodoptera frugiperda

“…O mutante R97A foi excluído da determinação desta faixa de amplitude de variação de kcat, pois foi demonstrado anteriormente que R97 modula através de interações não-covalentes o pKa do nucleófilo catalítico E399 (Marana et al, 2003). Logo, esta arginina tem papel essencial na catálise, sendo que os minúsculos valores de kcat observados para R97A sugerem que seu o papel catalítico é mais preponderante no efeito mutacional do que seu posicionamento como "hub" na rede estrutural.…”

“…Dados para este mutante estão na Tabela 7. (Wilkinson et al, 1983;Fersht, 1999 (Marana et al, 2003), quando foi demonstrado que R97 e Y331 modulam através de interações não-covalentes o pKa do nucleófilo catalítico E399. Logo, os resíduos R97 e Y331 tem papel essencial na catálise, além de serem "hubs" da rede estrutural de Sfgli.…”