In humans there are three subunits of enolase, α, β, and γ, each encoded by a separate gene that can combine to form five different isoenzymes: αα, αβ, αγ, ββ, and γγ.

[3][6] Recently, moonlighting functions of several enolases, such as interaction with plasminogen, have brought interest to the enzymes' catalytic loops and their structural diversity.

Enolase's Lys345 deprotonates the alpha hydrogen, and the resulting negative charge is stabilized by resonance to the carboxylate oxygen and by the magnesium ion cofactors.

In addition to coordination with the catalytic magnesium ions, the pKa of the substrate's alpha hydrogen is also lowered due to protonation of the phosphoryl group by His159 and its proximity to Arg374.

It has been inferred that levels of CSF neuron-specific enolase, serum NSE, and creatine kinase (type BB) are indicative in the prognostic assessment of cardiac arrest victims.

[20] Small-molecule inhibitors of enolase have been synthesized as chemical probes (substrate-analogues) of the catalytic mechanism of the enzyme and more recently, have been investigated as potential treatments for cancer and infectious diseases.

Attempts have been made to use this inhibitor as an anti-trypanosome drug,[25] and more recently, as an anti-cancer agent, specifically, in glioblastoma that are enolase-deficient due to homozygous deletion of the ENO1 gene as part of the 1p36 tumor suppressor locus (synthetic lethality).

[26] A natural product phosphonate antibiotic, SF2312 (CAS 107729-45-3), which is active against gram positive and negative bacteria especially under anaerobic conditions,[27] is a high potency inhibitor of Enolase 4zcw that binds in manner similar to phoshphonoacetohydroxamate 4za0.

[30] More recently, a derivative of SF2312, termed HEX, and a prodrug thereoff, POMHEX, were shown to exert anti-neoplastic activity against ENO1-deleted glioma in a pre-clinical intracranial orthotopic mouse model.

[33] Subsequent analysis using a commercial assay also indicated that ENOblock can inhibit enolase activity in biological contexts, such as cells and animal tissues.

[34] Active site transition state analogue Enolase inhibitors have been explored pre-clinically for the treatment of various microbial pathogens, as well as in precision oncology for tumors with 1p36 homozygous deletions, that lack ENO1.