Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization.
Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat 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 structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.
Angeles
T S
TS
IGEN, Inc., Rockville, Maryland 20852.
Smith
R G
RG
Darsley
M J
MJ
Sugasawara
R
R
Sanchez
R I
RI
Kenten
J
J
Schultz
P G
PG
Martin
M T
MT
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D006241:Haptens / Q000276:immunology; D006825:Hybridomas; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D006241:Haptens; D007135:Immunoglobulin Variable Region; D007527:Isoenzymes