TWO CONSERVED TRYPTOPHAN RESIDUES ARE RESPONSIBLE FOR INTRINSIC FLUORESCENCE ENHANCEMENT OF RUBISCO ACTIVASE UPON ATP BINDING

Authors: WANG, DAFU - UNIVERSITY OF ILLINOIS; PORTIS JR, ARCHIE

Submitted to: Photosynthesis Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2006
Publication Date: 5/5/2006
Citation: Wang, D., Portis Jr, A.R. 2006. Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement of Rubisco activase upon ATP binding. Photosynthesis Research. 88:185-193.

Interpretive Summary: The activity of Rubisco, the enzyme that captures carbon dioxide, often limits photosynthesis, the process by which plants use light energy from the sun to make carbohydrates for growth from carbon dioxide and water. Rubisco activity is maintained by another chloroplast protein, Rubisco activase. Rubisco activity might be increased to improve plant growth by altering its interaction with Rubisco activase. Past work showed that the intrinsic fluorescence of activase increases when it self-associates in preparation for interaction with Rubisco, providing an internal monitor of certain conformational changes. In this work, we identified the two amino acids that are responsible for the increase in fluorescence, which provides new insight into the nature of the conformational changes that occur. This information will benefit scientists attempting to modify the properties of Rubisco and the activase in ways beneficial for increased photosynthesis by crop plants.

Technical Abstract: Two species-invariant tryptophan residues at positions 109 and 250 were identified by site-directed mutagenesis of tobacco Rubisco activase as being responsible for the increase in intrinsic fluorescence of upon addition of ATP, which has been previously been attributed to an increased oligomerization of the protein. Substitution of W109, which is immediately prior to a “P-loop” sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the wildtype enzyme. In contrast, substitution of W250 with Tyr or Phe abolished 44% of increase in intrinsic fluorescence of activase with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The changes in the intrinsic fluorescence of W16 and W305 mutants upon addition of ATP were similar to those of the wild type, which indicates that W109 and W250 are the residues reporting the conformational change that increases the intrinsic of activase.