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Published on: November 23, 2019
The effect of p-hydroxymercuribenzoate and congeners on microsomal glucose-6-phosphatase
Abstract:
Iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2 mM. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate. 14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2-4 mumoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 X 10(-5) M p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3 mumoles 14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA greater than barbital greater than tryptophan greater than histidine greater than lysine greater than other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 X 10(-6) M. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.
Insights
p-hydroxymercuribenzoate (p-MB) inhibits microsomal glucose-6-phosphatase by altering enzyme conformation, not by reacting with sulfhydryls. This reversible inhibition is antagonized by certain compounds, suggesting a non-covalent interaction mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Membrane protein studies
Background:
- Microsomal glucose-6-phosphatase is a key enzyme in gluconeogenesis.
- Understanding its inhibition mechanisms is crucial for metabolic research.
- Sulfhydryl-reactive compounds are often used to probe enzyme active sites.
Purpose of the Study:
- To investigate the inhibitory effects of various sulfhydryl-reactive compounds on microsomal glucose-6-phosphatase.
- To elucidate the mechanism of inhibition by p-hydroxymercuribenzoate (p-MB).
- To determine if p-MB interacts with sulfhydryl groups or other amino acid residues.
Main Methods:
- Testing iodoacetamide, N-ethylmaleimide, p-MB, and HgCl2 as inhibitors.
- Using purified microsomal preparations (M2) and crude microsomes.
- Employing 14C-labelled p-MB for binding studies.
- Conducting kinetic analyses (double reciprocal plots) and dilution experiments.
- Assessing the effect of various compounds on p-MB inhibition.
Main Results:
- p-MB inhibited all tested preparations, with 50% inhibition at 5 x 10(-5) M on purified M2.
- Inhibition by p-MB was reversible by dithiothreitol, except after pre-incubation without substrate, which accelerated inactivation.
- Irreversible binding of 14C-p-MB to M2 protein was independent of mercurial concentration.
- Several compounds, notably EDTA and barbital, antagonized p-MB inhibition.
- p-MB increased Km and decreased Vmax for glucose-6-phosphate hydrolysis.
- HgCl2 was a more potent inhibitor than p-MB (Ki = 6 x 10(-6) M).
Conclusions:
- The inhibition of microsomal glucose-6-phosphatase by p-MB does not involve a reaction with sulfhydryl groups.
- p-MB likely alters enzyme conformation through interaction with other amino acid side chains.
- The reversible nature and competitive antagonism suggest a non-covalent binding mechanism for p-MB.
