2XH7

Engineering the enolase active site pocket: Crystal structure of the D321A mutant of yeast enolase 1

Summary for 2XH7

• Entry DOI: 10.2210/pdb2xh7/pdb
• Related: 1EBG 1EBH 1ELS 1L8P 1NEL 1ONE 1P43 1P48 2AL1 2AL2 2ONE 2XGZ 2XH0 2XH2 2XH4 3ENL 4ENL 5ENL 6ENL 7ENL
• Descriptor: ENOLASE 1, MAGNESIUM ION, 2-PHOSPHOGLYCERIC ACID, ... (4 entities in total)
• Functional Keywords: lyase, tim-barrel, enolase superfamily, glycolysis, gluconeogenesis, metal binding
• Biological source: SACCHAROMYCES CEREVISIAE (BAKER'S YEAST)
• Cellular location: Cytoplasm: P00924
• Total number of polymer chains: 2
• Total formula weight: 95904.85

Authors

Schreier, B.,Hocker, B. (deposition date: 2010-06-09, release date: 2010-08-25, Last modification date: 2023-12-20)

Primary citation

Schreier, B.,Hoecker, B.
Engineering the Enolase Magnesium II Binding Site -Implications for its Evolution.
Biochemistry, 49:7582-, 2010

PubMed Abstract: The glycolytic enzyme enolase catalyzes the reversible elimination of water from 2-phosphoglycerate (2-PGA) to form phosphoenolpyruvate (PEP). Two magnesium ions in the active site are thought to facilitate the reaction by activation of the C2 proton of 2-PGA and charge stabilization of the intermediate. The initial abstraction of a proton from a carboxylic acid is common to all members of the enolase superfamily, yet in all other known members of this superfamily, only one magnesium ion (MgI) per active site is sufficient to promote catalysis. We wanted to further investigate the importance of the second magnesium ion (MgII) for the catalytic mechanism of yeast enolase 1. Toward this end, we removed all MgII coordinating residues and replaced substrate-MgII interactions by introducing positively charged side chains. High-resolution crystal structures and activity assays show that the introduced positively charged side chains effectively prohibit MgII binding but fail to promote catalysis. We conclude that enolase is inactive without MgII, yet control mutants without additional positively charged side chains retain basal enolase activity through binding of magnesium to 2-PGA in an open active site without the help of MgII coordinating residues. Thus, we believe that ancestral enolase activity might have evolved in a member of the enolase superfamily that provides only the necessary catalytic residues and the binding site for MgI. Additionally, precatalytic binding of 2-PGA to the apo state of enolase was observed.

PubMed: 20690637
DOI: 10.1021/BI100954F

Experimental method

X-RAY DIFFRACTION (1.8 Å)