Substitutions for Gly-794 show that binding interactions are important determinants of the catalytic action of β-galactosidase (Escherichia coli)

Abstract

Substitutions of Gly-794 (beta-galactosidase) with Asp, Asn, Glu, and Lys caused decreased binding of substrates and inhibition by substrate analogs, while inhibition by planar and positively charged galactose analogs increased relative to the binding of substrates and the inhibition by substrate analogs. There was a correlation of the relative inhibition with the size of the substituted residue but no relationship to the presence or absence of a negative charge, and as the relative inhibition by the planar and positively charged galactose analogs increased, k3 (hydrolysis; degalactosylation) and kcat/Km (catalytic efficiency) values decreased. The k2 values (glycolytic cleavage; galactosylation) mainly increased for poor substrates (p-nitrophenyl beta-galactoside and lactose) but decreased for o-nitrophenyl beta-galactoside (a good substrate). Enzymes substituted with Asp or Asn were inhibited to a similar extent by planar and positively charged inhibitors and had similar effects on catalysis, while inhibition and catalytic effects on the enzyme substituted by Glu were quite different. If the negative charge was important, the Asp- and Glu-substituted enzymes should have been inhibited to a similar extent, while the Asn-substituted enzyme should have caused a different degree of inhibition. The enzyme substituted with a Lys at position 794 bound substrates and inhibitors very poorly, but the relative inhibition and the catalysis still correlated to size. Alterations of the size of the residue at position 794 cause modifications in the binding interactions and affected activity.(ABSTRACT TRUNCATED AT 250 WORDS)

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…The activity was not increased with all substrates-only those for which the first catalytic step (galactosylation) was rate determining. Further studies 57 showed that b-galactosidases with several different substitutions for Gly794 had similar properties. The structure of the enzyme 8 revealed that Gly794 is a ''hinge'' at one end of a loop near to the active site [ Fig.…”

Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by β-Galactosidase from Escherichia coli

“…This may be due to the larger size of serine/threonine derivatives leading to better interactions with the amino acid residues found in the large acceptor-binding site of the enzyme as compared to the smaller monosaccharides. However, these binding affinities were weaker than the experimental binding affinities of ONPG (−8.1~−8.2 kcal/mol) at the degalactosylation step [ 22 , 23 ]. This slightly weaker binding affinity of E. coli β-galactosidase towards its acceptors may also reflect the importance of the thermodynamic equilibrium between the transgalactosylation and hydrolysis reaction.…”

Distinct mechanisms of antibody‐mediated enzymatic reactivation in β‐galactosidase molecular sensors

“…In M278VP1, the acceptor site is a protruding loop extending towards the active site of the partner monomer, forming the activating interface between the components of the dimer [6]. In JX795A, the receiving, solvent‐exposed loop embraces the Gly 794 , an amino acid residue involved in substrate binding [28]. The atomic distances between each insertion site and both active sites in the corresponding dimer are shown in Table 2.…”