Enzymes and initial-rate investigations

What you’ll learn

• Explain enzyme specificity and reduction of activation energy.
• Interpret effects of temperature, pH and concentrations on rate.
• Calculate initial rates and identify suitable experimental controls.

Understand the idea

An enzyme is a biological catalyst whose active site interacts with particular substrates. Binding can induce a shape change that helps position reacting groups and stabilise the transition state, reducing activation energy. The enzyme is regenerated at the end of the reaction. Intracellular enzymes act within cells, whereas extracellular enzymes such as digestive enzymes act after secretion. Specificity depends on chemical interactions as well as the shape of the active site.

Rising temperature initially increases kinetic energy and the frequency of successful collisions. At sufficiently high temperatures, disruption of the protein’s stabilising interactions alters the active site and lowers activity. Changes in pH alter charges and interactions, potentially changing binding or folding. At a fixed enzyme concentration, increasing substrate concentration eventually produces a plateau because active sites are occupied much of the time. Increasing enzyme concentration can increase rate when sufficient substrate is available.

Core practical 4 compares initial rates while varying one of temperature, pH, enzyme concentration or substrate concentration. The initial rate is the tangent gradient near the start of a product–time curve, before substantial substrate depletion or product accumulation. Control the other relevant variables, use a buffer for pH, repeat measurements and compare variability. A fixed endpoint time can supply a relative rate through its reciprocal only when the amount of reaction required for the endpoint is constant.

Key formulae

$$\text{initial rate}=\left.\frac{\Delta\text{product}}{\Delta t}\right|_{\text{initial tangent}}$$

$$\text{relative rate}\propto\frac1{\text{time to a fixed endpoint}}$$

Worked example

An initial tangent to a product-volume graph passes through (0 s, 0 cm³) and (20 s, 6.0 cm³). Later, the curve becomes less steep. Calculate the initial rate and explain one reason for the slowing.

1. Use the tangent coordinates, rather than averaging the whole curve.
2. Initial rate is (6.0 − 0)/(20 − 0) = 0.30 cm³ per second.
3. As substrate is consumed, fewer substrate molecules may bind each second, lowering the rate.

Answer: 0.30 cm³ per second; substrate depletion can explain the later decrease.