ISOABZYMES - STRUCTURALLY AND MECHANISTICALLY SIMILAR CATALYTIC ANTIBODIES FROM THE SAME IMMUNIZATION

Mechanistic and structural comparisons of five catalytic monoclonal an tibodies generated from the same hybridoma fusion indicated that all f ive hydrolyze phenyl acetate by subtle variations of the same mechanis m. All of the antibodies showed a pre-steady-state multi-turnover burs t in which k(cat) and K(m) declined but k(cat)/K(m) did not change. Th e burst of one of the antibodies, 20G9, has previously been found to r esult from inhibition by the product, phenol. Although all of the anti bodies showed the burst, their individual values for k(cat), K(m), and hapten K(i) differed substantially. Three of the antibodies that were investigated for the effect of pH on k(cat) showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies . Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structu ral similarities, analysis of their sequences suggested that the antib odies are products of distinct, independent rearrangements of immunogl obulin gene segments that took place in different progenitor B cells. A plot of K(i) for hapten inhibition vs K(m)/k(cat) for substrate hydr olysis for the mechanistically related antibodies (''isoabzymes'') gav e a linear relationship suggesting a catalytic role for transition-sta te complementarity. Taken together with previous work [Martin et al.(1 991) Biochemistry 30, 9757-9761], the data conform to a mechanism in w hich the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.