Thiamine deficiency [TD] may result in significant dysfunction of nervous and cardiovascular systems. A reduction during TD in the activities of the thiamine pyrophosphate [TPP]-utilizing enzymes, transketolase [Tk], pyruvate dehydrogenase, and alpha-ketoglutarate dehydrogenase, has been implicated as the metabolic basis of tissue injury, but very little is known about the molecular mechanisms underlying these enzymatic changes. Our preliminary studies suggest that the rate of the TPP-dependent formation of holo-Tk, and perhaps of other TPP-utilizing enzymes as well, may be of fundamental importance in the pathogenesis of TD-related disorders. In order to study the molecular interactions between TPP-utilizing enzymes and their cofactor during assembly, we propose to first produce sufficient human Tk so that the crystallographic structure of Tk complexed with TPP can be determined. Also, key amino acid residues involved in TPP binding or in subunit-subunit interactions in the Tk holoenzyme will be specifically mutated to provide insight into how proteins bind to, and interact with TPP, and how they are assembled into a functional holoenzyme. Second, using a human cell culture system in which the thiamine antagonist, pyrithiamine, mimics increasing degrees of TD, we will systematically investigate the time course of inactivation of the three TPP-utilizing enzymes, and recovery of activity on changing to thiamine sufficient culture conditions. Potential effects on stability, synthesis rate, and/or assembly of the Tk holoenzyme will also be examined. The effects of thiamine on the expression of the genes encoding the TPP-utilizing enzymes will be studied by determining the mRNA levels for each enzyme during TD and thiamine sufficiency using this culture system. Finally, the effects of limited thiamine on TPP- utilizing enzymes will be studied in leukocytes of malnourished alcoholics before and after thiamine treatment. Lymphoblast cultures will also be established from these patients and others with demonstrated TD-related diseases in order to study the potential influence of interindividual differences in the rate of Tk holoenzyme formation on susceptibility to TD. Understanding the molecular mechanisms involved In thiamine utilization has important implications for the prevention, identification, and treatment of diverse TD-related disorders.
