STEP 2: Conversion of glucose-6-phosphate to fructose-6-phosphate- Phosphorylated sugar produced in step-1 is converted into the fructose-6-phosphate by the action of phospho-hexose isomerase .

STEP 3: Phosphorylation of fructose-6-phosphate- In this step, sugar is further phsophorylated at carbon 1 to produce fructose-1,6 bis phosphate by the action of Phosphofructokinase . In the phosphorylation reaction, phosphate ( γ-phosphate ) group of ATP is transferred to phosphorylated sugar to form fructose-1,6 bis phosphate. One molecule of ATP is utilized in this step.

STEP 4: Clevage of fructose 1,6-bis phosphate- This step is catalyzed by enzyme aldolase or fructose 1,6 bis aldolase to generate glyceraldehyde-3 phosphate (aldose) and dihydroxy acetone phosphate (ketose).

STEP 1-4: First 4 reactions of enzymatic conversion of glucose (6 carbon sugar) to glyceraldehydes-3 phosphate (aldose) and dihydroxy acetone phosphate (ketose) are considered as preparative phase of glycolysis and during this phase, two major events happen:

1. Commitment of Sugar for glycolysis- Phosphorylated products are negatively charged and impermeable to the cell membrane through passive diffusion. Glycolysis operates in cytosol and as a result first step of phosphorylation inhibits the passive movement of the particular glucose moiety and drive it to participate in further steps of glycolysis.

2. Activation of sugar- In the 1 st and 3 rd step of glycolysis, two phosphorylation reactions add potential energy into the molecule and hence activate the sugar to participate into the cleavage reaction to form two 3 carbon sugar moiety.

STEP 5: Interconversion of the triose phosphates- Three carbon sugar formed in step 4 undergoes internal conversion and as glyceraldehyde-3 phosphate can readily be able to enter into the next step, the ketose generated in step 4 is reversibly convereted into the glyceraldehydes-3 phosphate by triose-3-phosphate isomerase.

Figure 5.1: Different Reactions of Glycolysis.