How enzymes catalyze reactions and are regulated at the molecular level are fundamental questions in contemporary biology. We propose to address these questions by investigation of two highly regulated enzymes of carbohydrate metabolism, human brain hexokinase (HKI) and porcine liver fructose-1, 6- bisphosphatase (FBPase). HKI is believed to be the pacemaker of glycolysis in brain tissue, whereas FBPase is a crucial control step in gluconeogenesis. Thus these enzymes are related in the sense that one is involved in glucose utilization, whereas the other is critical to the synthesis of blood glucose. Brain which only comprises 2-3% of total mammalian body weight utilizes approximately 25% of the circulating blood glucose and about 20% of the oxygen consumed by the body. In cerebral tissue, under normal physiological conditions, glucose phosphorylation is mediated by HKI, and it is then metabolized via the Krebs cycle to synthesize ATP which is required in large quantities for brain metabolism. Human HKI was recently cloned and expressed in Escherichia coli in our laboratory. In order to gain insights into the putative glucose and ATP binding domains, the regulatory sites, and the N-terminal amino acid sequence responsible for HKI binding to the mitochondrial pore structure, studies involving site- specific mutagenesis will be undertaken. NMR spectroscopy will be used in conjunction with the mutagenesis experiments to investigate the mechanism of the HKI reaction, its molecular mechanism of regulation, and structure- function relationships of the enzyme. The 3-dimensional structure of FBPase is currently available. Analysis of this model can be used to evaluate the structure-function relationships that exist at the active and allosteric sites of this enzyme from site- specific mutagenesis studies. We will clone and express the pig kidney FBPase gene in E. coli as a prerequisite to doing the site-directed mutagenesis studies. Our goal will be to evaluate the kinetic and regulatory properties of the mutant and wild-type enzymes and to use this information to elucidate the structure-function relationships of liver FBPase.
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