Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center
Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate-(PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140,000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70,000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with (/sup 32/P)PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.
- OSTI ID:
- 5023380
- Journal Information:
- Biochemistry; (United States), Vol. 4
- Country of Publication:
- United States
- Language:
- English
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PHOSPHOTRANSFERASES
AMINO ACID SEQUENCE
BIOCHEMICAL REACTION KINETICS
PHOSPHORYLATION
ELECTROPHORESIS
GEL PERMEATION CHROMATOGRAPHY
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR WEIGHT
PEPTIDES
PHOSPHOENOLPYRUVATE
PHOSPHORUS 32
STREPTOCOCCUS
TRACER TECHNIQUES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
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DAYS LIVING RADIOISOTOPES
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KINETICS
LIGHT NUCLEI
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques