Enolase

is an enzyme that catalyzes a reaction of glycolysis. Glycolysis converts glucose into two 3-carbon molecules called pyruvate. The energy released during glycolysis is used to make ATP.^[1] Enolase is used to convert 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the 9th reaction of glycolysis: it is a reversible dehydration reaction.^[2]. Enolase is expressed abundantly in most cells and has been proven useful as a model to study mechanisms of enzyme action and structural analysis ^[3]. As with the reaction below, Enolase must have a divalent metal cation present to activate or deactivate the enzyme. The best cofactor would be Mg2+, but many, including Zn2+, Mn2+ and Co2+ can be used. The metal ion works by binding to the enzyme at the active site and producing a conformational change. This makes it possible for the substrate (2-PGA) to bind at the Enolase active site. Once this happens, a second metal ion comes in and binds to the enzyme to activate the Enolase catalytic ability.

Structure

The of enolase contains both alpha helices and beta sheets. The beta sheets are mainly parallel^[4]. As shown in the figure, enolase has about 36 alpha helices and 22 beta sheets (18 alpha helices and 11 beta sheets per domain). Enolase consists of two domains.

Structural Clasification of Proteins (SCOP)^[5]

Enolase is in the alpha and beta proteins class and has a fold of TIM beta/alpha-barrel. It comes from the Superfamily on Enolase C-terminal domain-like and is in the enolase family.

Mechanism

The of enolase as shown, involves Lys 345, Lys 396, Glu 168, Glu 211, and His 159. Enolase forms a complex with two at its active site. The substrate, 2PG, binds to the two . The Mg 2+ then forms a bond at the deprotonated carboxylic acid on the 1'C to connect it with enolase. It also is connects to Glu 211 and Lys 345. Glu 211 makes a hydrogen bond with the alcohol group on the 3'C. Lys 345 deprotonates the 2'C and then the 2'C forms an alkene with the 1'C which then moves the electrons forming the ketone onto the oxygen making it have a negative charge. The other oxygen, which already has a negative charge, then moves its electron to form a ketone with the 1'C. The electrons that made up the alkene between the 1'C adn 2'C then moves to form an alkene between the 2'C and 3'C. This breaks the bond with the alcohol on the 3'C which deprotonates Glu 211 on enolase to form H2O. Then the new molecule is released from enolase as PEP. PEP then goes on through another step in glycolysis to create pyruvate.

Fluoride ions inhibits glycolysis by forming a bond with Mg 2+ thus blocks the substrate (2PG) from binding to the active site of enolase.^[7]

Kinetics

Since Mg2+ is essential for binding the substrate, 2-PG, it is also needed at a specific quality in order to have a good rate, or velocity. The graph shows the V vs. [PGA], in which PGA is 2-PG, with two different concentrations of Mg2+. The upper curve, which also has greater Vmax, has an Mg2+ concentration of 10^-3 M while the lower curve, which has a lower Vmax, has an Mg2+ concentration of 10^-2 M^[9]. The Km is also larger the upper curve making the higher [Mg2+] more desirable.

Regulation

Enolase is found on the surface of a variety of eukaryotic cells as a strong plamingoen-binding receptor and on the surface of hematopietic celss such as monocytes, T cells and B cells, neuronal celss and endothelial cells. Enolase in muscle can bind other glycolytic enzymes, such as phosphoglycerate mutase, muscle creatine kinase, pyruvate kinase, and muscle troponin, with high affinity. This suggests that they make a functional glycolytic segment in the muscle where ATP production is required in order for the muscle to contract. Myc-binding protein (MBP-1) is similar to the a-enolse structure and is found in the nucleus as a DNA-binding protein^[10]. Enolase is regulated by the concentration of Mg2+ and the previous steps of glycolysis.

Other interesting information

Enolase is present in all tissues and organisms with the ability to do glycolysis or fermentation. Recent studies have Enolase concentration samples in order to determine certain conditions and their severity. For instance, high concentrations of Enolase in cerebrospinal fluid (CSF) are more strongly associated with astrocytoma than other enzymes like aldolase, pyruvate kinase, and creatine kinase. High concentrations of Enolase in the CSF are also linked to the fastest rate of tumor growth and increased chances of heart attack or stroke. Enolase can be competitively inhibited by fluoride for the substrate 2-PGA. In drinking water with added fluorination, oral bacteria Enolase activity is inhibited without harmed humans. This works to prevent cavities.

3D structures of enolase

Update January 2013

Enolase

3dip, 2qgy – ENO – unidentified
1oep - TbENO – Trypanosoma brucei
2ptw – TbENO (mutant)
2ptx - TbENO (mutant) + sulfate
2pty - TbENO (mutant) + PEP
2ptz, 2pu0, 2pu1 - TbENO (mutant) + PAH
2pa6 – ENO – Methanocaldococcus jannaschii
1w6t – ENO – Streptococcus pneumoniae
1iyx – ENO – Enterococcus hirae
1pdy, 1pdz – ENO – European lobster
3qn3 – ENO – Campylobacter jejuni
3tqp – ENO – Coxiella burnetii
3ugv – ENO – Alpha proteobacterium
4a3r – BsENO – Bacillus subtilis
4h1z – ENO – Rhizobium meliloti

Enolase 1

3b97, 2psn – hENO1 - human
3otr – ENO1 – Toxoplasma gondii
3qtp – ENO1 – Entamoeba histolytica
1e9i - EcENO1 – Escherichia coli
2xgz, 2xh0, 2xh2, 2xh4, 2xh7 – EcENO1 residues 2-437 (mutant)
3uj2 – ENO1 – Anaerostipes caccae
3h8a, 2fym – EcENO1 + RNase E
3enl, 4enl – yENO1 - yeast
2al1, 2al2, 1ebh – yENO1 + Mg
1p43, 1p48 – yENO1 (mutant)
1ebg, 1els - yENO1 + PAH
1l8p - yENO1 (mutant) + PAH
2one, 1one – yENO1 + PEP + phosphoglycerate
5enl, 7enl - yENO1 + phosphoglycerate
1nel, 6enl – yENO1 + inhibitor

Enolase 2

1te6 – hENO2
2akm, 2akz – hENO2 + inhibitor
4ewj – ENO2 – Streptococcus suis

Enolase 3

2xsx, 3ucc, 3ucd, 3uje, 3ujf, 3ujr, 3ujs - hENO3

2, 3-diketo-5-methylthiopentyl-1-phosphate enolase

2zvi – DK-MTP-1-P BsENO
2oej - BkDK-MTP-1-P ENO + Pi – Geobacillus kaustophilus
2oek, 2oel - BkDK-MTP-1-P ENO + ion
2oem - BkDK-MTP-1-P ENO + Mg + phosphate derivative

Additional Resources

For additional information, see: Carbohydrate Metabolism