Substrate (chemistry)

For other uses, see Substrate.

In chemistry, the term substrate is highly context-dependent. Broadly speaking, it can refer either to a chemical species being observed in a chemical reaction, or to a surface on which other chemical reactions or microscopy are performed.

In the former sense, a reagent is added to the substrate to generate a product through a chemical reaction. The term is used in a similar sense in synthetic and organic chemistry, where the substrate is the chemical of interest that is being modified. In biochemistry, an enzyme substrate is the material upon which an enzyme acts. When referring to Le Chatelier's principle, the substrate is the reagent whose concentration is changed.

Spontaneous reaction

S → P

• Where S is substrate and P is product.

Catalysed reaction

S + C → P + C

• Where S is substrate, P is product and C is catalyst.

In the latter sense, it may refer to a surface on which other chemical reactions are performed or play a supporting role in a variety of spectroscopic and microscopic techniques, as discussed in the first few subsections below.

Substrate Investigations

Investigation examined how changing the temperature of a full cream milk, 0.05% sodium bicarbonate and lipase solution affects the rate of enzyme activity, measured by change in pH using Sparkvue pH probe. Lipase catalyses breakdown of triglycerides into fatty acids, lowering the solution’s pH, providing indirect measure of enzyme activity. As temperature increases, enzyme and substrate molecules gain kinetic energy, increasing frequency of successful collisions and enzyme-substrate complex formation. Activity maximised at an optimal temperature, but beyond this point, enzymes begin to denature as bonds in their structure break, altering shape of the active site and reducing catalytic function. Independent variable was temperature (°C), and dependent variable was rate of pH change. Controlled variables included substrate volume and enzyme concentration. A control group not required, as removing temperature would eliminate the thermal conditions necessary for enzyme function. Investigation aimed to determine the temperature at which lipase activity is most efficient.

School-Based Methodologies

Controlled experiment: The method isolates temperature as the independent variable while keeping substrate and enzyme concentrations constant, ensuring a fair test. pH was chosen as indirect but quantifiable measure of lipase activity, as the breakdown of lipids produces fatty acids that lower pH, allowing enzyme activity to be tracked reliably over time.

Five millilitres of full cream milk were combined with three drops of 10% lipase and 0.05% sodium bicarbonate solution. The mixture was placed in a 0°C ice water bath for five minutes, then removed and tested with three drops of indicator; pH was measured using a Sparkvue pH probe. This procedure was repeated at 20°C, 40°C, and 60°C to compare the effect of temperature on enzyme activity.

Statement Trial

Greatest pH decrease occurred at 20°C, indicating this temperature enabled the highest lipase activity and most efficient enzyme function under the experimental conditions.

Enzyme Summarisation

Results demonstrated that lipase exhibited its highest catalytic efficiency at 20°C, where greatest pH decrease (–7.6%) was observed, indicating most rapid production of fatty acids. At 40°C, enzyme activity markedly reduced, with minimal pH drop (–2.1%), while at 0°C and 60°C, pH increased (+5.6% and +2.2% respectively), suggesting at 0°C, enzyme activity was inhibited due to reduced molecular motion and limited enzyme-substrate collisions, while at 60°C, activity was lost due to denaturation from thermal disruption of enzyme’s tertiary structure. Overall, the data formed non-standard shape, with peak reaction rate at 20°C and substantially lower activity at temperatures above and below this point.

E + S ⇌ ES → EP ⇌ E + P

• Where E is enzyme, S is substrate, and P is product

While the first (binding) and third (unbinding) steps are, in general, reversible, the middle step may be irreversible (as in the rennin and catalase reactions just mentioned) or reversible (e.g. many reactions in the glycolysis metabolic pathway).

By increasing the substrate concentration, the rate of reaction will increase due to the likelihood that the number of enzyme-substrate complexes will increase; this occurs until the enzyme concentration becomes the limiting factor.

Substrate promiscuity

Main article: Enzyme promiscuity

Aim was investigate how temperature affects lipase activity, measured by pH change. Hypothesis predicted highest activity near 37°C. However, results refuted this: peak activity occurred at 20°C, with the greatest pH decrease (–7.6%), indicating most fatty acid production. At 0°C and 60°C, pH increased (+5.6%, +2.2%) and 40°C dropped slightly (–2.1%). Temperature affected the pH by altering kinetic energy and enzyme-substrate complex formation. The data formed non-standard trend, with peak enzyme activity at 20°C and minimal pH change at other temperatures, resulting in inverted V-shape rather than typical enzyme activity curve. 0°C and 60°C considered outliers due to unexpected pH increases. Experiment lacked precision due to absence of repeats, which prevented evaluation of variability. Accuracy was reduced by use of uncalibrated pH probes; closer results are to true value, more accurate they are - calibration improves this. Validity compromised by pH increases that did not align with expected enzyme behaviour. Experiment was repeatable, but not reproducible due to limited procedural detail. Improvements include replicates, freshly prepared lipase, calibrated probes, and thermostatically controlled incubation (respectively).

Sensitivity

Sensitive substrates, also known as sensitive index substrates, are drugs that demonstrate an increase in AUC of ≥5-fold with strong index inhibitors of a given metabolic pathway in clinical drug-drug interaction (DDI) studies.

Moderate sensitive substrates are drugs that demonstrate an increase in AUC of ≥2 to <5-fold with strong index inhibitors of a given metabolic pathway in clinical DDI studies.

Interaction between substrates

Metabolism by the same cytochrome P450 isozyme can result in several clinically significant drug-drug interactions.

Active Sites and Optimisation

Investigation examined how changing the temperature of full cream milk, 0.05% sodium bicarbonate and lipase solution affects rate of enzyme activity, measured by change in pH using Sparkvue pH probe. Results of investigation refute hypothesis, which predicted lipase activity be greatest near 37°C. Instead, highest enzyme activity occurred at 20°C, indicated by greatest pH decrease (–7.6%), reflecting maximal fatty acid production. Suggesting, under the experimental conditions, lipase functioned most efficiently at lower-than-expected temperature, with activity substantially reduced or absent at both higher and lower temperatures.