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Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function

John E. Wilson

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA

View larger version (16K): [in a new window]   Fig. 1. Phosphorylation, catalyzed by hexokinase, is the initial step in common pathways of Glc metabolism.

View larger version (16K): [in a new window]   Fig. 2. Glucose (Glc) phosphorylation to glucose-6-phosphate (Glc-6-P) by mitochondrially bound hexokinase with exogenous ATP or with ATP generated by oxidative phosphorylation. The rate of Glc phosphorylation was determined at equivalent [ATP], either added exogenously in the absence of oxidative phosphorylation (triangles) or generated by oxidative phosphorylation (circles). From either source, the [ATP] was subsaturating, with the rate of Glc phosphorylation well below that seen when the ATP levels were acutely raised by addition of saturating levels of exogenous ATP (squares). Reprinted with permission from BeltrandelRio and Wilson (1991).

View larger version (16K): [in a new window]   Fig. 3. Glucose (Glc) phosphorylation by mitochondrially bound hexokinase, with ATP generated by oxidative phosphorylation in the presence of increasing concentrations of exogenous ATP. ATP production by oxidative phosphorylation was initiated by addition of ADP at the indicated time. Glc phosphorylation was coupled to NADPH production, monitored by absorbance at 340 nm (A), in the presence of excess glucose-6-phosphate (Glc-6-P) dehydrogenase. The concentrations of exogenous ATP, present at the time of ADP addition, were 1.1, 0.66, 0.22 and 0 mmol l-1 for curves A–D, respectively. Note that the steady state attained was independent of the original extramitochondrial [ATP]. Reprinted with permission from BeltrandelRio and Wilson (1992b).

View larger version (12K): [in a new window]   Fig. 4. Effect of adding excess unlabeled Pi on the 32P/14C ratio of glucose-6-phosphate (Glc-6-P) formed by mitochondrially bound hexokinase or nonmitochondrially bound yeast hexokinase. Oxidative phosphorylation was initiated with 32Pi present as substrate for oxidative phosphorylation, and [14C]Glc as substrate for hexokinase. At 3 min, excess 31Pi was added, reducing the specific activity of ATP subsequently produced by oxidative phosphorylation. This resulted in a precipitous decrease in the 32P/14C ratio of Glc-6-P formed by yeast hexokinase (squares) using extramitochondrial ATP as substrate, but a much slower decrease in the 32P/14C ratio of Glc-6-P produced by mitochondrially bound hexokinase (open circles). Filled circles, total Glc-6-P produced by mitochondrially bound hexokinase. Reprinted with permission from de Cerqueira Cesar and Wilson (1995).

View larger version (24K): [in a new window]   Fig. 5. Schematic representation of the experimental strategy for comparing utilization of extramitochondrial ATP by mitochondrially bound hexokinase or nonbound yeast hexokinase. (A) Mitochondrially bound hexokinase (HK) is represented at the center of the panel, with additional mitochondria, containing little or no bound hexokinase, shown in more peripheral regions. For the latter, rat brain mitochondria that had been depleted of hexokinase by treatment with glucose-6-phosphate, which causes release of the mitochondrially bound hexokinase, were used in earlier experiments. Later experiments, however, used rat liver mitochondria which, as isolated, do not contain bound hexokinase. Extramitochondrial ATP is distributed throughout the extramitochondrial space. (B) Analogous situation, but with an equivalent amount of nonmitochondrially bound yeast hexokinase (YHK) in place of the mitochondrially bound hexokinase. The basic strategy is to determine the rate of glucose phosphorylation by a fixed amount of bound or nonbound hexokinase as the rate of extramitochondrial ATP production is increased by addition of increasing numbers of mitochondria devoid of bound hexokinase. Reprinted with permission from de Cerqueira Cesar and Wilson (2002).

View larger version (11K): [in a new window]   Fig. 6. Rate of glucose (Glc) phosphorylation by mitochondrially bound and nonbound hexokinase, with increasing rates of ATP production from oxidative phosphorylation. The rate of Glc phosphorylation () is expressed relative to the maximal rate of phosphorylation (), the latter determined with saturating levels of exogenous ATP in the absence of oxidative phosphorylation. The rate of Glc-6-P production by nonmitochondrially bound yeast hexokinase (circles) is closely correlated with the rate of ATP production. In contrast, the rate of Glc phosphorylation by mitochondrially bound hexokinase (squares) is insensitive to increasing levels of extramitochondrial ATP produced by non-hexokinase-bearing mitochondria, consistent with the view that the mitochondrially bound enzyme is restricted to intramitochondrial ATP, produced by the mitochondria to which the enzyme is bound, as substrate. Reprinted with permission from de Cerqueira Cesar and Wilson (1998).

View larger version (13K): [in a new window]   Fig. 7. Inhibition of mitochondrially bound hexokinase by the glucose-6-phosphate analog, 1,5-anhydroglucitol-6-P (1,5-AnG6P), with intramitochondrially generated (open circles) or extramitochondrial (filled circles) ATP as substrate. Reprinted with permission from Hashimoto and Wilson (2000).

View larger version (9K): [in a new window]   Fig. 8. Coordination of glycolytic and oxidative phases of glucose (Glc) metabolism. The rate of Glc phosphorylation by mitochondrially bound hexokinase, using intramitochondrially generated ATP as substrate, is correlated with the rate of oxidative phosphorylation. This mechanism is suggested to ensure coordination of Glc phosphorylation, the initial step in glycolytic metabolism, with terminal oxidative stages (tricarboxylic acid cycle, with associated electron transport and oxidative phosphorylation; bold curved arrows) occurring in the mitochondria, avoiding the buildup of potentially toxic lactate.