Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: Differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose

J. Roitelman, I. Shechter

Research output: Contribution to journalArticlepeer-review

Abstract

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Ki(app) = 21.7 μM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979 Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Ki(app) = 550 μM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 μM and 53 μM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Ki(app) = 50 μM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 μM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Ki(app) = 175 μM in the presence of 300 μM NADH). These results support a proposal that thiol-dependent reduction of enzyme disulfide(s) or binding of an allosteric activator to it cause conformational changes in the microsomal reductase protein that alter the properties of its catalytic NADPH-binding site(s).

Original languageEnglish
Pages (from-to)828-835
Number of pages8
JournalJournal of Lipid Research
Volume27
Issue number8
StatePublished - 1986

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