In plants, animals and bacteria the glycine cleavage system catalyzes the following reversible reaction:
:  Glycine + Hfolate + NAD  ↔ 5,10-methylene-Hfolate + CO + NH + NADH + H
In the enzymatic reaction, H-protein activates the P-protein, which catalyzes the decarboxylation of glycine and attaches the intermediate molecule to the H-protein to be shuttled to the T-protein. The H-protein forms a complex with the T-protein that uses tetrahydrofolate and yields ammonia and 5,10-methylenetetrahydrofolate. After interaction with the T-protein, the H-protein is left with two fully reduced thiol groups in the lipoate group. The glycine protein system is regenerated when the H-protein is oxidized to regenerate the disulfide bond in the active site by interaction with the L-protein, which reduces NAD to NADH and H.
When coupled to serine hydroxymethyltransferase, the glycine cleavage system overall reaction becomes:
: 2 glycine + NAD + HO → serine + CO + NH + NADH + H
In humans and most vertebrates, the glycine cleavage system is part of the most prominent glycine and serine catabolism pathway. This is due in large part to the formation 5,10-methylenetetrahydrofolate, which is one of the few C donors in biosynthesis. In this case the methyl group derived from the catabolism of glycine can be transferred to other key molecules such as purines and methionine.
This reaction, and by extension the glycine cleavage system, is required for photorespiration in C plants. The glycine cleavage system takes glycine, which is created from an unwanted byproduct of the Calvin cycle, and converts it to serine which can reenter the cycle. The ammonia generated by the glycine cleavage system, is assimilated by the Glutamine synthetase-Glutamine oxoglutarate aminotransferase cycle but costs the cell one ATP and one NADPH. The upside is that one CO is produced for every two O that are mistakenly taken up by the cell, generating some value in an otherwise energy depleting cycle. Together the proteins involved in these reactions comprise about half the proteins in mitochondria from spinach and pea leaves. The glycine cleavage system is constantly present in the leaves of plants, but in small amounts until they are exposed to light. During peak photosynthesis, the concentration of the glycine cleavage system increases ten-fold.
In the anaerobic bacteria, Clostridium acidiurici, the glycine cleavage system runs mostly in the direction of glycine synthesis. While glycine synthesis through the cleavage system is possible due to the reversibility of the overall reaction, it is not readily seen in animals.