Laser excitation studies of the product release steps in the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase

J Biol Chem. 2007 Nov 2;282(44):32015-20. doi: 10.1074/jbc.M706098200. Epub 2007 Sep 11.

Abstract

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 approximately 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.

MeSH terms

  • Catalysis
  • Cyanobacteria / enzymology*
  • Cyanobacteria / metabolism
  • Kinetics
  • Lasers
  • NADP / metabolism
  • Oxidoreductases Acting on CH-CH Group Donors / chemistry*
  • Oxidoreductases Acting on CH-CH Group Donors / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Thermodynamics

Substances

  • Recombinant Proteins
  • NADP
  • Oxidoreductases Acting on CH-CH Group Donors
  • protochlorophyllide reductase