The isozymes have 87-88% identical amino acid sequence identity and though they are structurally and functionally similar, they have different localizations: PGK2, encoded by an autosomal gene, is unique to meiotic and postmeiotic spermatogenic cells, while PGK1, encoded on the X-chromosome, is ubiquitously expressed in all cells.

[1] In the Calvin cycle in photosynthetic organisms, PGK catalyzes the phosphorylation of 3-PG, producing 1,3-BPG and ADP, as part of the reactions that regenerate ribulose-1,5-bisphosphate.

PGK has been reported to exhibit thiol reductase activity on plasmin, leading to angiostatin formation, which inhibits angiogenesis and tumor growth.

[9] The bivalent metal assists the enzyme ligands in shielding the bound phosphate group's negative charges, allowing the nucleophilic attack to occur; this charge-stabilization is a typical characteristic of phosphotransfer reaction.

The PGK-catalyzed transfer of the phosphate group from 1,3-BPG to ADP to yield ATP can power[clarification needed] the carbon-oxidation reaction of the previous glycolytic step (converting glyceraldehyde 3-phosphate to 3-phosphoglycerate).

[citation needed] The enzyme is activated by low concentrations of various multivalent anions, such as pyrophosphate, sulfate, phosphate, and citrate.

[18] Since the trait is X-linked, it is usually fully expressed in males, who have one X chromosome; affected females are typically asymptomatic.

[17][2] On a molecular level, the mutation in Pgk1 impairs the thermal stability and inhibits the catalytic activity of the enzyme.

[3] Due to its wide specificity towards nucleotide substrates, PGK is known to participate in the phosphorylation and activation of HIV antiretroviral drugs, which are nucleotide-based.