THE IMMUNOLOGICAL EVOLUTION OF CATALYSIS

The germline genes used by the mouse to generate the esterolytic antib ody 48G7 were cloned and expressed in an effort to increase our unders tanding of the detailed molecular mechanisms by which the immune syste m evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transitio n state analogue by a factor of 10(4), primarily the result of a decre ase in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with sub strate, k(cat)/K-m, from 1.7 x 10(2) M(-1) min(-1) to 1.4 x 10(4) M(-1 ) min-l The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that non e of the nine residues in which somatic mutations have been fixed dire ctly contact the hapten. Thus, in the case of 48G7, affinity maturatio n appears to play a conformational role, either in reorganizing the ac tive site geometry or limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic a nd directed active site mutants underscore the role of transition stat e stabilization in the evolution of this catalytic antibody.