Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 282, Issue 44, Pages 32015-32020Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M706098200
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Funding
- BBSRC [BB/D01963X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D01963X/1] Funding Source: researchfish
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The latter stages of the catalytic cycle of the light- driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6- ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP(+) and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP(+) is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP(+) concentration, suggesting that conformational changes are required prior to NADP(+) release. Following NADP(+) release, the NADPH rebinds to the enzyme with a maximum rate constant of similar to 72 s(-1). At elevated temperatures (> 298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.
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