Healthy cellular activity is dependent on the proper folding, 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, with the consequent production of unique products of catalysis. This study is thus directed at two major objectives: (1) the solving of many long standing issues of RubisCO function through the incorporation of procedures of random mutagenesis and genetic selection, coupled to functional enzymological approaches; (2) the use of unique and newly discovered microbial RubisCO molecules to facilitate an understanding of enzyme function. These objectives present novel and fresh approaches to key questions of structure, function, folding and assembly that have long been at issue. Indeed, RubisCO is a major example of a protein where X-ray structural studies have not provided answers as to the basis of enzyme efficiency or substrate selectivity. Thus, it is paramount that the new approaches become incorporated into investigations of this protein so that we may gain an understanding as to how this single protein (RubisCO) controls the course of metabolism by favoring one catalytic function over another. The combined microbiological/genetic/biochemical focus of this study will thus greatly enhance available X-ray structural models and will provide many new insights relative to this important protein's function and unique ability to control many aspects of metabolism. Of particular interest will be the relationship of protein folding and catalysis to cellular function.
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