Subunits of multimeric enzymes have evolved interactive protein domains by which assembly and activity are determined. Ribulose- 1,5-bisphosphate carboxylase/oxygenase (rubisco), with its two subunits, LSU and SSU, is an excellent model for studying such subunit assembly and interactions. Extensive biochemical and physiological studies are available, including protein/gene sequences and crystallographic structures. Rubisco SSU, which does not contribute any active site residues, is essential in the L8S8 rubisco holoenzyme for activity. This project will focus on the functions of domains in SSU in determining i) folding of SSU, ii) assembly of active L8S8, and iii) SSU influence of kinetic parameters. Two of these aspects have been extensively studied by us (Wasmann et al., 1989; Ramage, 1990; Smrcka et all, 1990a,b) providing the basis for specific mutations which we will introduce into SSU by site directed mutagenesis. After site- directed mutagenesis, SSU genes will be expressed in bacteria and assembled with soluble cyanobacterial L8 in vitro. Assembly of modified SSU will also be investigated following transport into chloroplast. Kinetics of activation, stability of substrate and effector binding, and binding parameters of substrate analogs will be measured with holoenzymes composed of homologous and heterologous subunits. Similar studies on higher plant rubisco are prevented as LSU is extremely insoluble in the absence of SSU. Therefore, altered SSU will be expressed and assembled in vitro, in isolated chloroplasts, and in vivo, following inactivation of resident SSU expression by a ribozyme gene construct. (As an intermediary step to establishing the functionality of ribozymes, ribozyme constructs and mutated SSU will be tested in vitro, after electroporation of genes into mesophyll protoplasts). The effect of modified SSU on several parameters of activity of higher plant rubisco will be determined. The hypothesis is that SSU (cyanobacteria and in higher plants) compensates conformational changes induced by the binding of substrate to the active site.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9017419
Program Officer
Marcia Steinberg
Project Start
Project End
Budget Start
1991-02-01
Budget End
1994-07-31
Support Year
Fiscal Year
1990
Total Cost
$261,000
Indirect Cost
Name
University of Arizona
Department
Type
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721