Class I proteins form a protonated Schiff base intermediate linking a highly conserved active site lysine with the DHAP carbonyl carbon.

Additionally, tyrosine residues are crucial to this mechanism in acting as stabilizing hydrogen acceptors.

Two histidine residues in the first half of the sequence of these homologs have been shown to be involved in binding zinc.

[1] The protein subunits of both classes each have an α/β domain folded into a TIM barrel containing the active site.

The chief products of the Calvin cycle are triose phosphate (TP), which is a mixture of DHAP and G3P, and fructose 6-phosphate.

The aldolase used by plants and algae in the Calvin cycle is usually a plastid-targeted protein encoded by a nuclear gene.

Aldolase catalyzes and also Aldolase is used in the reversible trunk of gluconeogenesis/glycolysis Aldolase is also used in the part of the Calvin cycle shared with gluconeogenesis, with the irreversible phosphate hydrolysis at the end catalyzed by fructose 1,6-bisphosphatase In gluconeogenesis 3-PG is produced by enolase and phosphoglycerate mutase acting in series In the Calvin cycle 3-PG is produced by RuBisCO G3P is produced by phosphoglycerate kinase acting in series with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in gluconeogenesis, and in series with glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) in the Calvin cycle Triose-phosphate isomerase maintains DHAP and G3P in near equilibrium, producing the mixture called triose phosphate (TP) Thus both DHAP and G3P are available to aldolase.

Aldolase has also been implicated in many "moonlighting" or non-catalytic functions, based upon its binding affinity for many other proteins including F-actin, α-tubulin, light chain dynein, WASP, Band 3 anion exchanger, phospholipase D (PLD2), glucose transporter GLUT4, inositol trisphosphate, V-ATPase and ARNO (a guanine nucleotide exchange factor of ARF6).

These associations are thought to be predominantly involved in cellular structure, however, involvement in endocytosis, parasite invasion, cytoskeleton rearrangement, cell motility, membrane protein trafficking and recycling, signal transduction and tissue compartmentalization have been explored.