Title

Regulation of Bacillus stearothermophilus phosphoglycerate mutase by pH and manganese

Date of Completion

January 2000

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Phosphoglycerate mutase (PGM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate during carbohydrate metabolism and are of two types—those that require 2,3-diphosphoglycerate as a cofactor and those that do not. Here I have shown that PGMs from endospore-forming bacteria including Bacillus, Clostridium and Sporosarcina species are 2,3-diphosphoglycerate-independent, but absolutely require Mn 2+ ions. At low concentrations of Mn2+ these PGMs are exquisitely sensitive to pH so that a pH change from 8 to 6 causes a several-hundred fold inhibition in activity. The pH-sensitivity of PGMs from endospore-formers is significant for their regulation in vivo because the pH of the spore protoplast decreases from a value of ∼8.1 to ∼6.5 during sporulation, and this acidification is believed to cause the inhibition of PGM leading to the accumulation of 3-phosphoglycerate in spores which serves as an energy source at germination. ^ Binding studies show that B. stearothermophilus PGM binds 1 Mn2+ ion with a Kd of 50 μM at pH 6 and 2 Mn2+ ions at pH 8 with Kds of 1 μM and 70 μM. PGM functions optimally with 2 Mn2+ ions at pH 8 and the reduced affinity for these essential ions at pH 6 combined with a decrease in the Vmax of catalysis causes a drastic reduction in PGM activity at acidic pH. ^ The crystal structure of B. stearothermophilus PGM described here shows that the Mn2+ ions keep active site residues optimally positioned to bind substrate and are also involved in the catalytic reaction. The pH-dependence of Mn2+ binding is due to the ionization states of the 3 histidines that coordinate the Mn2+ ions; these residues are presumably protonated at pH 6 and are therefore unable to efficiently ligate Mn2+. This structure is also the first one described for a 2,3-diphosphoglycerate-independent type PGM and has allowed elucidation of the catalytic mechanism which is believed to proceed via a phosphoenzyme intermediate. The importance of the Mn2+ ions and key active site residues has been confirmed by analysis of site-directed mutants. ^