A rapid quench kinetic study of ribulosebisphosphate carboxylase and in vitro modelling of resonant energy transfer between chlorophyll A solvated species in water/acetone solvent systems
Abstract
The net rate constants for conversion of substrate to intermediates to product of the ribulosebisphosphate carboxylase enzyme of Rhodospirillum rubrum were determined by fitting the equations corresponding to a sequential irreversible reaction to the levels of components from rapid kinetic quenches. The first intermediate of the reaction, the ene-diol of 1,5-ribulosebisphosphate, was detected as the monophosphate elimination product obtained upon acid quenching the enzymic reaction mixture and reduction of the products with sodium borohydride. Similarly, the second intermediate, carboxy-keto-arabinitolbisphosphate (CKABP), was detected as a mixture of 2$\sp\prime$- and 4-carboxyarabinitol 1,5-bisphosphate. Remaining substrate, ribulosebisphosphate (RuBP), was determined as a mixture of ribitol and arabinitol bisphosphate. The rate constants of RuBP consumption and conversion of ene-diol to CKABP exhibited saturation kinetics with respect to bicarbonate concentration. In order for these results to accommodate the published mechanism of this enzyme, a step which becomes rate limiting at high carbon dioxide concentrations would be required between the formation of the ene-diol and the carboxylation. Additionally, a revised mechanism for in vivo carboxylation from the analysis of early publications of in vivo carboxylation is presented. Chlorophyll A fluorescence quenching, dependent upon increasing water content of solvent or increasing chlorophyll A concentration is shown to fit a theoretical expression of dipole-dipole transfer between acetone-solvates of chlorophyll A and the dihydrate dimer species. This fit is corroborated by a 680 nm peak present in the difference absorption spectrum obtained upon the conversion of the acetone solvate species to the long-wavelength absorbing species attributable to the polymeric chlorophyll A species. There is also nearly complete overlap present between the donor, acetone-solvate species, absorption spectrum and the putative acceptor species, dihydrate dimer, required for dipole-dipole transfer of energy.
Degree
Ph.D.
Advisors
Low, Purdue University.
Subject Area
Biochemistry
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