Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.
Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.
Miyashita
H
H
Protein Engineering Research Institute, Suita, Osaka, Japan.
Hara
T
T
Tanimura
R
R
Fukuyama
S
S
Cagnon
C
C
Kohara
A
A
Fujii
I
I
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000235:genetics; D002701:Chloramphenicol / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D004952:Esters / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011355:Prodrugs / Q000378:metabolism
D017151:Antibodies, Catalytic; D004952:Esters; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011355:Prodrugs; C055966:immunoglobulin Fv; D006639:Histidine; D002701:Chloramphenicol