It has been a major objective of the research carried out in my laboratory during the past 10 years to gain a better understanding of carbon dioxide fixation in animal, plant and microbial cells. The long- range goals include the characterization of the biologically important carboxylases and the elucidation of their catalytic and regulatory mechanisms. These carboxylase-catalyzed reactions serve diverse metabolic functions ranging from fatty acid biosynthesis, amino acid and propionate catabolism, gluconeogenesis, and urea formation to photosynthesis. Despite these differences in metabolic function, there are basic chemical similarities among the enzymatic carboxylations, e.g., the mechanisms of carboxylation of phosphoenolpyruvate (PEP) and biotin. Work in our laboratory has included investigations on both the "biotin" enzymes, i.e., acetyl CoA carboxylase, propionyl CoA carboxylase and methylmalonyl CoA-pyruvate transcarboxylase, and the "non-biotin" carboxylases, i.e., phosphoenolpyruvate, (PEP) carboxykinase, PEP carboxylase, and ribulose diphosphate carboxylase. During the initial period of the present Research Grant, substantial progress was made in the investigation of the latter-mentioned group of "non-biotin" carboxylases. Purification methods developed in our laboratory permit the large-scale preparation of homogeneous PEPCK and RuDPC and of nearly homogeneous PEPC which will be necessary for the research projected. Furthermore, as will be described in more detail in a later section, the molecular characteristics of all 3 carboxylases have been investigated in some detail. Together with the kinetic analyses and studies on the reaction mechanisms already carried out by our group, an excellent foundation is provided for the work proposed.