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

Substitutions of Gly-794 (β-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 β-galactoside and lactose) but decreased for o-nitrophenyl β-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. If planar and positively charged galactose derivatives are transition state analogs, they must mimic the transition state for galactosylation (k2) more than the transition state for degalactosylation (k3), since k2 usually increased when the relative inhibition by these inhibitors was better while k3 always decreased. The amounts of Mg2+ required for activation of the substituted enzymes did not correlate with either charge or size.Key words: β-galactosidase, binding, glycine, aspartic, asparagine, glutamic, lysine, mechanism, transition state, catalysis, site-specific substitution.