Related Experiment Videos
The enzymatic activity of phosphoglycerate mutase from gram-positive endospore-forming bacteria requires Mn2+ and is
M Chander1, B Setlow, P Setlow
1Department of Biochemistry, University of Connecticut Health Center, Farmington 06030, USA.
Abstract:
The enzymatic activity of phosphoglycerate mutase (Pgm) from three gram-positive endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, and Sporosarcina ureae) requires Mn2+ and is very sensitive to pH; at low concentrations of Mn2+, a pH change from 8 to 6 resulted in greater than 30- to 200-fold decreases in the activity of these Pgms. However, Pgm deactivation at pH 6 was reversed by shifting the enzyme to pH 7 or 8. Free Mn2+ was not directly involved in Pgm catalysis, although enzyme-bound Mn2+ may be involved. The rate of catalysis by Mn(2+)-containing Pgm was also slightly pH dependent, although the Km for 3-phosphoglyceric acid appeared to be the same at pH 6, 7, and 8. These findings suggest that Mn2+ binds to catalytically inactive Pgm and converts it to a catalytically competent form, and further, that pH influences the efficiency with which the enzyme binds Mn2+. The extreme pH sensitivity of the Mn(2+)-dependent Pgms supports a model in which this enzyme is inhibited during sporulation by acidification of the forespore, thus allowing accumulation of the spore's large depot of 3-phosphoglyceric acid. The activity of Pgm from two closely related gram-positive bacteria that do not form spores (Planococcus citreus and Staphylococcus saprophyticus) also requires Mn2+ and is pH sensitive. In contrast, the Pgm activities from two more distantly related non-endospore-forming gram-positive bacteria (Micrococcus luteus and Streptomyces coelicolor) are neither dependent on metal ions nor particularly sensitive to pH.
Insights
Phosphoglycerate mutase (Pgm) activity in spore-forming bacteria requires manganese (Mn2+) and is highly pH-sensitive. This pH sensitivity may explain Pgm inhibition during bacterial sporulation.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Phosphoglycerate mutase (Pgm) is a key enzyme in glycolysis.
- Endospore-forming bacteria utilize Pgm, but its regulation during sporulation is not fully understood.
- Manganese (Mn2+) is known to be essential for the activity of some enzymes.
Purpose of the Study:
- To investigate the role of manganese (Mn2+) and pH on the activity of phosphoglycerate mutase (Pgm) from various gram-positive bacteria.
- To explore the potential implications of Pgm's pH sensitivity in bacterial sporulation.
Main Methods:
- Enzyme activity assays were performed on Pgm isolated from different bacterial species.
- The effect of varying Mn2+ concentrations and pH levels on Pgm activity was measured.
- Kinetic parameters, including Km, were determined under different pH conditions.
Main Results:
- Pgm from endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, Sporosarcina ureae) requires Mn2+ and exhibits extreme pH sensitivity.
- Pgm deactivation at low pH (6) was reversible by returning to neutral or alkaline pH (7-8).
- Mn2+ appears to bind to inactive Pgm, converting it to an active form, with pH influencing binding efficiency.
Conclusions:
- The pH sensitivity of Mn2+-dependent Pgm suggests a regulatory mechanism during bacterial sporulation, where forespore acidification inhibits the enzyme.
- This inhibition allows for the accumulation of 3-phosphoglyceric acid, a key component of the spore's energy reserves.
- Pgm from non-spore-forming bacteria shows varying degrees of Mn2+ and pH dependence, correlating with their phylogenetic relationship to spore-formers.