Three thiol groups are important for the activity of the liver microsomal glucose-6-phosphatase system. Unusual behavior of one thiol located in the glucose-6-phosphate translocase.
Liver microsomal glucose-6-phosphatase (Glc-6-Pase) is a multicomponent system involving both substrate and product carriers and a catalytic subunit. We have investigated the inhibitory effect of N-ethylmaleimide (NEM), a rather specific sulfhydryl reagent, on rat liver Glc-6-Pase activity. Three thiol groups are important for Glc-6-Pase system activity. Two of them are located in the glucose-6-phosphate (Glc-6-P) translocase, and one is located in the catalytic subunit. The other transporters (phosphate and glucose) are not affected by NEM treatment. The NEM alkylation of the catalytic subunit sulfhydryl residue is prevented by preincubating the disrupted microsomes with saturating concentrations of substrate or product. This suggests either that the modified cysteine is located in the protein active site or that substrate binding hides the thiol group via a conformational change in the enzyme structure. Two other thiols important for the Glc-6-Pase system activity are located in the Glc-6-P translocase and are more reactive than the one located in the catalytic subunit. The study of the NEM inhibition of the translocase has provided evidence of the existence of two distinct areas in the protein that can behave independently, with conformational changes occurring during Glc-6-P binding to the transporter. The recent cloning of a human putative Glc-6-P carrier exhibiting homologies with bacterial phosphoester transporters, such as Escherichia coli UhpT (a Glc-6-P translocase), is compatible with the fact that two cysteine residues are important for the bacterial Glc-6-P transport.
Clottes
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Department of Obstetrics and Gynaecology, Ninewells Hospital and Medical School, Dundee University, Dundee, DD1 9SY, Scotland.
Burchell
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D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D012542:Scattering, Radiation; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014638:Vanadates / Q000494:pharmacology
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D010710:Phosphates; D013438:Sulfhydryl Compounds; D014638:Vanadates; D019298:Glucose-6-Phosphate; D005952:Glucose-6-Phosphatase; D005947:Glucose; D003545:Cysteine; D005033:Ethylmaleimide