Kinetic studies of the brain hexokinase reaction.

The mechanism of the hexokinase reaction has been the subject of numerous investigations in recent years (l-5). ijgren and Engstrijm reported the incorporation of Pa from labeled adenosine triphosphate (ATP) into the phosphoserine moiety of hexokinase in the absence of substrate glucose and concluded that an enzyme-phosphate intermediate participated in the reaction (2). Gamble and Najjar, however, were unable to demonstrate a glucose-glucose 6-phosphate exchange with yeast hexokinase in the absence of nucleotide substrates (3). The latter investigators did observed labeling of hexokinase by Cl4 glucose in the absence of ATP (4) and suggested that an enzyme-glucose intermediate participated in the reaction. Their postulation appeared to explain adequately many of those data available from studies of the phosphotransferase. A series of recent studies appeared to be at variance with both of the above mentioned hypotheses (5). In 1951 it was reported that glucose 6-phosphate severely inhibited mammalian hexokinase, probably noncompetitively (6). This contention was supported by investigations of Crane and Sols (7). The results of these studies implied that glucose and its phosphorylated product occupy separate enzymatic sites (6, 7). A similar view has recently been advanced regarding dehydrogenases (8) ; however, this explanation may not be valid (9). Hexokinase from yeast does not appear to be as susceptible to glucose 6-phosphate inhibition as the mammalian enzyme (10, 11). Adenosine diphosphate (ADP) (12) and certain sugars (13) have been reported to be competitive inhibitors of hexokinase. Unfortunately, most of the investigations regarding inhibition were essentially preliminary. The purpose of the present study was to investigate the mechanism of the hexokinase reaction kinetically. It will be shown that these investigations do support a mechanism in which the product of the first substrate dissociates from the enzyme before addition of the second substrate. These data appear to be consistent with the proposals that either an enzyme-phosphate or an enzyme-glucose intermediate participates in the hexokinase reaction (2, 4).