SUBSTITUTIONS FOR GLY-794 SHOW THAT BINDING INTERACTIONS ARE IMPORTANT DETERMINANTS OF THE CATALYTIC ACTION OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI)

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 inhibi tion by substrate analogs. There was a correlation of the relative inh ibition with the size of the substituted residue but no relationship t o the presence or absence of a negative charge, and as the relative in hibition by the planar and positively charged galactose analogs increa sed, k(3) (hydrolysis; degalactosylation) and k(cat)/K-m (catalytic ef ficiency) values decreased. The k(2) values (glycolytic cleavage; gala ctosylation) mainly increased for poor substrates (p-nitrophenyl beta- galactoside and lactose) but decreased for o-nitrophenyl beta-galactos ide (a good substrate). Enzymes substituted with Asp or Asn were inhib ited to a similar extent by planar and positively charged inhibitors a nd had similar effects on catalysis, while inhibition and catalytic ef fects on the enzyme substituted by Glu were quite different. If the ne gative charge was important, the Asp- and Glu-substituted enzymes shou ld 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 inhibitor s very poorly, but the relative inhibition and the catalysis still cor related to size. Alterations of the size of the residue at position 79 4 cause modifications in the binding interactions and affected activit y. If planar and positively charged galactose derivatives are transiti on state analogs, they must mimic the transition state for galactosyla tion (k(2)) more than the transition state for degalactosylation (k(3) ), since k(2) usually increased when the relative inhibition by these inhibitors was better while k(3) always decreased. The amounts of Mg2 required for activation of the substituted enzymes did not correlate with either charge or size.