Healthy cellular activity is dependent on the proper assembly and subsequent catalytic function of key enzymes of intermediary metabolism. Our primary interest and long-term goal is to understand factors which regulate the process by which carbon dioxide is assimilated into organic compounds of the cell, a process which is common to all organisms. Primary carbon dioxide assimilation is catalyzed by the enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO). This protein also has the capacity to function as an internal monooxygenase and catalyzes the oxygenolytic cleavage of RuBP under suitable conditions. RubisCO is thus a bifunctional protein in which the same polypeptide chain catalyzes the first step of two competing reactions of cellular metabolism; indeed recent studies indicate that this unique enzyme may function as an isomerase, an epimerase, and a phosphatase as well. This study is thus directed at three major areas: (1) the mechanism by which chaperonin proteins influence the folding of large (L) and small (S) subunits of L/8S/8 RubisCO; (2) the molecular basis by which RubisCO is able to discriminate between its two gaseous substrates, carbon dioxide and oxygen; and (3) the process by which the activity of RubisCO is regulated in the cell via posttranslational modification and the use of additional proteins. A system has been established for the RubisCO folding studies that will greatly enhance recent results indicating the importance of factor(s) other than known chaperonin proteins. In addition, novel selection procedures have been developed to enhance the RubisCO substrate specificity studies; thus residues and regions that influence catalysis and substrate specificity may be elucidated at a level beyond prejudicial site-directed mutagenesis procedures. Such studies will nicely enhance X-ray and structural models that have already been provided for several RubisCOs. These studies thus present an excellent opportunity to relate control of cellular metabolism to the proper folding and function of an important, yet complex oligomeric protein.
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