Substrate-level phosphorylation in glycolysis is a process where ATP is produced directly in the enzymatic reactions of glycolysis. It occurs when a phosphate group is transferred from a phosphorylated intermediate directly to ADP, forming ATP. This happens twice in glycolysis: once during the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate, and again when phosphoenolpyruvate is converted to pyruvate. In contrast, oxidative phosphorylation is a process that occurs in the mitochondria during aerobic respiration. It involves the transfer of electrons from NADH and FADH2 through the electron transport chain, creating a proton gradient across the mitochondrial membrane. This gradient drives the synthesis of ATP by ATP synthase. While substrate-level phosphorylation generates ATP directly and independently of any external electron acceptors, oxidative phosphorylation relies on a chain of redox reactions and the establishment of a proton gradient. Substrate-level phosphorylation is thus a more immediate but less efficient way of producing ATP compared to the high-yield process of oxidative phosphorylation.

Fluctuations in ATP and AMP levels play a critical role in regulating glycolysis, primarily through allosteric effects on key enzymes. High levels of ATP indicate that the cell has sufficient energy, leading to the inhibition of phosphofructokinase, a major regulatory enzyme in glycolysis. This inhibition slows down glycolysis, preventing excessive production of ATP when it is not needed. Conversely, high levels of AMP signal low energy availability in the cell. AMP activates phosphofructokinase, thereby accelerating glycolysis to produce more ATP. This regulation ensures that glycolysis responds dynamically to the cell's energy needs, ramping up when energy supplies are low and slowing down when energy is abundant. Such a feedback mechanism is crucial for maintaining cellular energy homeostasis, allowing cells to efficiently adapt their metabolic rates in response to changing energy demands. This adaptive response is key to cellular survival, particularly in fluctuating environmental conditions.

The conversion of NAD+ to NADH in glycolysis is a pivotal step in cellular respiration. This conversion occurs during the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, where NAD+ accepts electrons and protons to become NADH. NADH holds high-energy electrons, which are vital for the later stages of cellular respiration, particularly in the electron transport chain. In aerobic respiration, NADH donates these electrons to the electron transport chain in the mitochondria, leading to a series of reactions that ultimately generate a significant amount of ATP through oxidative phosphorylation. This process also regenerates NAD+, allowing glycolysis to continue. In anaerobic conditions, where the electron transport chain is inactive, NADH is used to regenerate NAD+ in processes like lactic acid fermentation or alcoholic fermentation, ensuring the continuation of glycolysis. Therefore, the conversion of NAD+ to NADH not only facilitates energy capture in glycolysis but also links glycolysis to the broader context of cellular respiration.

The investment phase in glycolysis is significant because it prepares the glucose molecule for energy extraction. During this phase, two molecules of ATP are expended to phosphorylate glucose and convert it into a form that can be split into two three-carbon sugars. This phosphorylation is crucial as it traps glucose within the cell and reduces the activation energy required for subsequent steps. The investment of ATP upfront is essential as it leads to a greater yield of ATP in the later stages of glycolysis. Additionally, the conversion of glucose to fructose-1,6-bisphosphate, which is then split into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, is a key step in diversifying the metabolic pathways. These three-carbon molecules can enter not only glycolysis but other metabolic pathways as well, illustrating the interconnected nature of cellular metabolism. Therefore, the investment phase, despite the initial consumption of ATP, sets the stage for a more efficient and versatile energy extraction process.

Hexokinase, the enzyme catalysing the first step of glycolysis, is crucial for several reasons. It phosphorylates glucose to form glucose-6-phosphate, a reaction that commits glucose to the glycolytic pathway. This phosphorylation is vital as it prevents glucose from leaving the cell and increases its reactivity, facilitating subsequent metabolic reactions. Hexokinase is tightly regulated to maintain cellular energy balance. It is inhibited by its product, glucose-6-phosphate, a classic example of feedback inhibition. This inhibition prevents over-accumulation of glucose-6-phosphate and ensures that glycolysis proceeds only when glucose levels are sufficiently high and the cell requires energy. Additionally, hexokinase has a high affinity for glucose, allowing it to function effectively even at low glucose concentrations. This characteristic is particularly important in maintaining energy production in cells under varying glucose availability, highlighting hexokinase's role in ensuring metabolic flexibility and efficiency.