Doctoral Dissertations

Date of Award

12-1983

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Fred C. Hartman

Committee Members

Salil Niyogi, Frank Kenney, Bruce Jacobson, Ray Popp, Ed Bernstine

Abstract

Ribulosebisphosphate carboxylase/oxygenase catalyzes both the carboxylation and oxidative cleavage of ribulose 1,5-bisphosphate, opposing reactions in photosynthetic carbon assimilation and photorespiration, respectively. One approach to understanding this bifunctionality entails identification of active-site regions of the enzyme by affinity labeling. Comparison of active-site residues in enzymes from evolutionarily distant should reveal conserved molecular features which are likely to sources function in catalysis.

Irregularities in a published report from another laboratory prompted the reexamination of the binding site for pyridoxal 5'-phosphate in the carboxylase from Rhodospirillum rubrum, described in Part I. A single pyridoxal 5'phosphate-labeled peptide was obtained. The reactive lysyl residue lies in a seven-residue sequence common to all known carboxylases.

Part II describes the synthesis and characterization of four potential affinity labels for the carboxylase. Reductive amination of ribulose 1,5-bisphosphate with bifunctional amines by cyanoborohydride and bromoacetylation of the isolated amino bisphosphates gave 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate (reagents A and B, epimeric at C2), 2-[2-(bromoacetamido)ethyl]amino-2-deoxypentitol 1,5- bisphophate (reagent C), 2-[4-(bromoacetamido)butyl]amino-2-deoxypentitol 1,5-bisphosphate (reagent D), and 2-{4-[4-(bromoacetamido)phenyl methyl]- anilino}-2-deoxypentitol 1,5-bisphosphate (reagent E).

The interactions of the epimeric reagents A and B with the carboxylase consistent with the documented effect of configuration at C2 on Reductive amination of were binding to the enzyme, Of the two, only reagent B Irreversibly inactivated the R. rubrum enzyme in a time-dependent manner, of reagent B-modified tryptic peptides revealed two approximately equal sites of labeling, His44 and Cys58 both of which were subject to protection by 2-carboxyribitol 1,5-bisphosphate. Although these residues are not conserved in the six known carboxylase sequences, they lie in a Isolation region of homology not previously known to be at or near the active site of the enzyme.

Reagents C and D also fulfilled criteria of affinity labeling; the former alkylated Met335 while the latter alkylated His44, Cys55, and Met335. The methionyl residue in the R. rubrum enzyme, previously modified by two affinity labels, is replaced by leucine in the other carboxylase. Reagent E, although a competitive inhibitor, exhibited no time-dependent effect on carboxylase activity. The implications of these findings to the active-site geometry of the carboxylase are discussed

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