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The mechanism of phosphoglucomutase from Micrococcus lysodeikticus
Abstract:
The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the (32)P label but no induced transport of the (14)C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the K(m) for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with (14)C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3x10(6)s(-1). Consistent with these findings is the fact that (14)C label exchanged between the substrates twice as rapidly as the (32)P label at chemical equilibrium. Further, since the (14)C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.
Insights
The phosphoglucomutase enzyme mechanism in Micrococcus lysodeikticus was clarified. Contrary to prior beliefs, its phosphoenzyme mechanism mirrors that of rabbit muscle phosphoglucomutase, involving rapid phosphoenzyme isomerization.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Phosphoglucomutase (PGM) catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate.
- Understanding the precise catalytic mechanism of PGM is crucial for metabolic pathway analysis.
Purpose of the Study:
- To elucidate the catalytic mechanism of phosphoglucomutase from Micrococcus lysodeikticus.
- To determine if the mechanism aligns with known PGM enzymes, such as the rabbit muscle isoform.
Main Methods:
- Utilized induced-transport assays with radiolabeled substrates ((32)P and (14)C).
- Investigated enzyme kinetics at varying substrate concentrations (0.15mM, 1mM, 26mM).
- Analyzed substrate and intermediate exchange rates at chemical equilibrium.
Main Results:
- Low substrate concentrations supported a phosphoenzyme mechanism with rapid phosphoenzyme isomerization.
- Results excluded intramolecular phosphate transfer and sequential mechanisms with lower K(m) values.
- High substrate concentrations indicated phosphoenzyme isomerization rate constants exceeding 3x10^6 s(-1).
- Glucose-1,6-diphosphate was not an obligatory intermediate.
Conclusions:
- The mechanism of Micrococcus lysodeikticus phosphoglucomutase is fundamentally similar to the rabbit muscle enzyme.
- A novel mechanism is proposed to explain rapid phosphoenzyme isomerization without formal isomerization steps.