Competitive inhibition of human brain hexokinase by metrizamide and related compounds

We have compared the competitive inhibitory effects of 2-deoxyglucose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2-[3-acetamido-2,4,6-triiodo-5-(N-methylacetamido) benzamido]-2-deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with Kis of 2.8 and 2.5 mM, respectively, for the mitochondrial and soluble forms. Glucosamine exhibited Kis of 0.58 and 0.29 mM, while 2-deoxyglucose exhibited Kis of 0.074 and 0.15 mM and N-acetylglucosamine 0.098 and 0.092 mM for these two forms, respectively. N-Benzoylglucosamine was by far the most effective inhibitor tested, with Ki values of 0.0086 and 0.022 mM, respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N-acetylglucosamine, 2-deoxyglucose, and N-benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2-deoxyglucose, N-acetylglucosamine, and N-benzoylglucosamine. Since N-benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N-benzoylglucosamine. However, gas chromatography-mass spectrometry analysis of metrizamide did not indicate the presence of N-benzoyl-glucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze-dried eluate fractions localized the inhibitory effect to the metrizamide peak.