Subtle active-sit alterations of ribulosebisphosphate carboxylase/oxygenase through concerted application of site-directed mutagenesis and chemical modification

Author

Harry Bache Smith

Date of Award

12-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Fred C. Hartman

Committee Members

Frank Kenney, Audrey Stevens, Salil Niyogi

Abstract

Four amino acid residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum (viz. Glu48, Lysl66, Lysl91, and Lys329) have been previously assigned to the active site through chemical, mutagenesis, and sequence studies. In order to define their catalytic functions more fully, and to ascertain their possible roles in influencing substrate specificity, subtle structural perturbations have been introduced into each of the pertinent side chains through a combination of site-directed mutagenesis —specifically, replacement of the pertinent residue with cysteine— and subsequent chemical modification. In this way, carboxymethylation of the Glu48-+Cys protein effectively lengthens the glutamyl side chain by insertion of a sulfur atom between the β- and γ-methylene groups, while aminoethylation of the Lysl66→Cys, Lysl91→Cys, or Lys329→Cys protein entails the mere replacement of a given 7-methylene group by a sulfur atom. Such structural changes are considerably more subtle than previous probes of the active site, and are intended to minimize the possibility of conformational disruption of the active site so that specific structure/function correlations may be drawn with confidence. While substitution of any of the four native residues with cysteine obliterates catalytic activity, the subsequent, appropriate chemical modification partially restores enzyme activity: Treatment of the Glu48→Cys protein with iodoacetate restores 3-4% of the wild-type carboxylase activity. Incubation of the Lysl66→Cys and Lys329→Cys proteins with bromoethylamine results in novel enzymes characterized by k_cal values which are, respectively, 20% and 60% of the wild-type value; ethylene imine similarly restores to the Lysl91→Cys protein a k_cal that is 4-7% of the wild-type value.

The significant impairment of k_cal that accompanies slight structural alterations of each of the four side chains underscores the stringency of the requirement of the corresponding wild-type residue for optimal catalysis. The extent to which catalytic competence is preserved through each alteration is nevertheless significant, so that the substrate specificity associated with each novel active site can be assessed. Despite the active-site modifications implemented at each of the three native lysyl positions, evidence for an altered CO₂/O₂ specificity is lacking. Substitution of the glutamyl residue at position 48 with carboxymethylcysteine, however, exerts a profound, fivefold reduction in the enzyme's specificity factor. This latter finding represents the first example of a major change in substrate specificity effected by structural alteration of an active-site side chain.

Recommended Citation

Smith, Harry Bache, "Subtle active-sit alterations of ribulosebisphosphate carboxylase/oxygenase through concerted application of site-directed mutagenesis and chemical modification. " PhD diss., University of Tennessee, 1989.
https://trace.tennessee.edu/utk_graddiss/11538