We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation. The sequence information was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes. Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function. The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain. Sequence comparisons suggest that the 50-kDa mammalian and yeast glucokinases arose separately in evolution. In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencing of the coding regions for other HKs and related proteins.