Mammalian glucokinase and its gene.

Mammalian glucokinase was identified 30 years ago as a distinct form of hexokinase in rat liver. The hexokinases (ATP: hexose 6phosphotransferases, EC 2.7.1.1) constitute a family of evolutionarily and stucturally related enzymes present in eukaryotic cells from yeast to mammals. In the cells of higher organisms, the physiologically significant substrate for these enzymes is Dglucose. The reaction catalysed by the hexokinases, ATP + Dglucose -+ ADP+ D-glucose 6-phosphate, is the first and obligatory step for glucose utilization after transport of the sugar into the cell. Mammalian tissues contain four different hexokinases which can be isolated by conventional protein separation techniques and for which cDNAs have been cloned. The isoenzymes of the rat have been designated hexokinases type I-IV or A-D in order of increasing negative net charge. The subject of this Review is hexokinase type IV or D, usually called glucokinase. Glucokinase stands apart from all the other hexokinases by a number of criteria. The first and most striking is its low affinity for glucose. The enzyme is half-saturated with glucose at 6 mM, compared with Km values in the micromolar range for the three other mammalian hexokinases. This feature led to the discovery of the enzyme and underlies its key role in the physiology of glucose homeostasis. The second hallmark ofmammalian glucokinase is its highly typical tissue distribution. The glucokinase gene is transcribed and the mRNA translated into active enzyme only in hepatocytes and insulin-secreting f-cells of the pancreatic islets of Langerhans, reflecting the great functional specialization of this isoenzyme. A third outstanding feature is the developmental and multihormonal regulation of the enzyme, illustrated most dramatically by the transcriptional induction of the glucokinase gene by insulin in the liver. The distinctive kinetics of glucokinase, its tissue-specific expression and its hormonal regulation were recognized within a few years of the discovery of the enzyme. However, our understanding of these particular characteristics has remained superficial until recently. The main reason for limited progress was the difficulty of purifying the enzyme, hence of raising specific antibodies or obtaining peptide sequence for the isolation of cDNA clones. Once this obstacle was surmounted, studies on glucokinase became a very fertile field of research. The most recent and medically rewarding outcome of this research has been the discovery of mutations of the glucokinase gene as the cause of one subtype of non-insulin-dependent diabetes mellitus (NIDDM). The purpose of this article is to review the recent developments on the structure and function of the glucokinase gene and its gene products, as they relate to our understanding of blood glucose homeostasis. The reader interested in historical perspectives and a complete background on the biochemistry of glucokinase should refer to the classical reviews of Walker [1], Weinhouse [2] and Colowick [3]. Recent commentaries on topical aspects are also available [4-8]. The glucokinase gene was first cloned from the rat and the structure of the gene in this species can serve as the standard of reference (Figure 1 a). The most remarkable feature is the presence of alternative promoters, responsible for the initiation of transcription at different sites on the DNA in hepatic and endocrine cells. The first clue to the existence of cell-type-specific promoters came from the sequences oftwo quasi-full-length cDNAs isolated from rat liver and insulinoma libraries [9,10]. The sequences were essentially identical for more than 2000 nucleotides starting from the 3' ends of the cDNAs, but segments of approximately 100 nucleotides at the 5' ends were found to differ entirely. The 5' specific sequences ofthe cDNAs were mapped to widely separated sites in genomic DNA by Magnuson and co-workers [10,11]. These investigators further identified nine exons, numbered 2-10 in the transcription unit, whose assembly gives rise to the common sequence found in the liver and insulinoma-derived cDNAs. The leader exon encoding the 5' end of the hepatic mRNA, termed exon 1L, was contained in a phage A clone which also carried the common exons 2-4. The leader exon for the 5' end of the insulinoma mRNA, termed exon 1lf in reference to the fl-cells of the islets of Langerhans, was localized in a different phage clone with non-overlapping genomic DNA. It was therefore concluded that the liver-specific exon IL was contiguous to the body of the structural gene, whereas the islet-type exon Ifi was located at an unspecified distance further upstream. The intervening sequence between the two leader exons has yet to be mapped accurately. Several attempts to isolate rat genomic DNA clones for the entire region have remained unsuccessful in my laboratory, perhaps suggesting unusual features of this DNA. In any event, more than 22 kb ofDNA separate the two leader exons in the rat gene (Figure la). The fact that the upstream exon lfl is used exclusively in isletderived cells, and the downstream exon 1L exclusively in liver, was established by primer extension and nuclease protection experiments and further confirmed by reverse transcription and PCR [10]. It should be noted that the two tissue-specific exons 1 of the glucokinase gene specify not only the 5' untranslated regions of the islet and liver mRNAs, but also their initial 45 nucleotides of protein coding sequence. It follows that the rat islet and liver glucokinase enzymes will differ in primary structure by 15 amino acids (including initiator Met) at the N-terminal ends of the molecules, for a total sequence of 465 amino acid residues. In addition to the differential splicing ofleader exons associated with the cell-specific control of transcription initiation, other modes of alternative splicing are known to affect glucokinase transcripts. An optional cassette exon has been identified in the rat gene between the originally described exons 1 L and 2 (Figure la). This cassette exon, termed exon 2A, is retained in a minor fraction of glucokinase mRNA in rat liver [12]. Alternative